Methods and compositions for treating cancer by targeting the clec2d-klrb1 pathway

ABSTRACT

Provided are methods and compositions for treating cancer in a subject in need thereof. One of the top gene products in glioblastoma multiforme (GBM) is KLRB1 (also known as CD161), a C-type lectin protein that binds to CLEC2D. Binding of CLEC2D to the KLRB1 receptor inhibits the cytotoxic function of NK cells as well as cytokine secretion. KLRB1 is only expressed by small subpopulations of human blood T cells, and consequently little is known about the function of this receptor in T cells. However, preliminary data demonstrate that KLRB1 expression is induced in T cells within the GBM microenvironment. In an exemplary embodiment, a method is provided comprising administering an agent capable of blocking the interaction of KLRB1 with its ligand. The agent may comprise an antibody or fragment thereof, which may bind KLRB1 or CLEC2D.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/585,422, filed Nov. 13, 2017. The entire contents of theabove-identified application are hereby fully incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. CA173750and Grant No. CA202820 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (BROD-2355WP.ST25.txt”;Size is 8 Kilobytes and it was created on Nov. 13, 2018) is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The subject matter disclosed herein is generally directed tocompositions and methods for treating cancer.

BACKGROUND

Glioblastoma multiforme (GBM) is one of the most challenging humancancers. It is the most aggressive type of brain cancer, and there is noclear way to prevent the disease.

Over the past few years novel immunosuppressive mechanisms have beenidentified in GBM patients, with a primary focus on scRNA-seq analysisof T cells directly isolated from surgically resected lesions. One ofthe top gene products identified is KLRB1 (also known as CD161), aC-type lectin protein that binds to CLEC2D. Binding of CLEC2D to theKLRB1 receptor inhibits the cytotoxic function of NK cells as well ascytokine secretion. KLRB1 is only expressed by small subpopulations ofhuman blood T cells, and consequently little is known about the functionof this receptor in T cells. However, preliminary data demonstrate thatKLRB1 expression is induced in T cells within the GBM microenvironment.An immunohistochemistry study demonstrated that CLEC2D (also calledLLT1) is expressed by human gliomas, with expression increasing with WHOgrade of malignancy. In contrast, there was little labeling with aCLEC2D specific antibody in sections from normal human brain tissue.These conclusions are supported by an analysis of TCGA RNA-seq dataperformed in collaboration with Dr. Shirley Liu (DFCI) whichdemonstrated significantly increased expression of CLEC2D in GBMcompared to normal brain tissue (p=5.1×10⁻¹¹). This analysis alsohighlighted increased expression of CLEC2D relative to the correspondingnormal tissue in many other cancer types, including all types of renalcancer (p<2×10⁻¹⁶ for KIRC), lung adenocarcinoma (p=5.5×10⁻¹¹), colonadenocarcinoma (p=3.1×10⁻¹²) and other cancers. Accordingly, it seemsthat KLRB1 functions as an inhibitory receptor for human T cells bybinding to the CLEC2D ligand on tumor cells. This hypothesis issupported by preliminary data from a humanized mouse model of GBM whichdemonstrate that inactivation of the KLRB1 gene in primary human T cellsgreatly enhances their cytotoxic function within tumors.

SUMMARY

In one aspect, the invention provides a method of treating a diseasecharacterized by increased expression of killer cell lecting likereceptor (KLRB1) in immune cells, comprising administering to a subjectin need thereof one or more agents in an amount sufficient to either:(i) block binding of CD161 to one or more CD161 ligands; (ii) reduceexpression of KLRB1 (the gene encoding CD161); (iii) reduce expressionof one or more CD161 ligands (iv) block binding of CLEC2D to a receptorof CLEC2D other than CD161, or any combination thereof.

In some embodiments, the one or more agents may comprise an antibody, orfragment thereof, that binds CD161. The one or more agents may comprisean antibody, or fragment thereof, that binds to the one or more CD161ligands. Alternatively, the method comprises administering a solubleCD161 protein, or fragment thereof, that binds to one or more of theCD161 ligands. In some embodiments, the antibody may be a humanized orchimeric antibody.

In some embodiments, reducing expression of KLRB1, or expression of oneor more CD161 ligands, comprises administering a programmable nucleicacid modifying agent configured to reduce expression of KLRB1, or reduceexpression of one or more CD161 ligands.

In some embodiments, the programmable nucleic acid modifying agent maybe a CRISPR-Cas, a zinc finger, a TALE, or a meganuclease. TheCRISPR-Cas may be a CRISP-Cas9, a CRISPR-Cas12, a CRISPR-Cas13, or aCRISPR-Cas14.

In some embodiments, the disease is cancer. The cancer may becharacterized by increased expression of a CD161 ligand by cancer cellsor other cells in the tumor microenvironment. Alternatively, one or moreimmune cell types in the tumor microenvironment may be characterized byincreased expression of KLRB1. In some embodiments, the one or moreCD161 ligands comprises CLEC2D.

In some embodiments, the one or more immune cells are tumor infiltratinglymphocytes (TILs).

In some embodiments, the cancer is a glioblastoma, melanoma, livercancer, renal cancel, lung adenocarcinoma, or colon adenocarcinoma.

In some embodiments, the one or more agents may be administered in acombination treatment regimen comprising checkpoint blockade therapy,vaccines, targeted therapies, radiation therapy, chemotherapy, and/oradoptive cell therapy (ACT).

The checkpoint blockade therapy may comprise anti-PD-1, anti-CTLA4,anti-TIM-3 and/or anti-LAG3.

The vaccine may be a neoantigen vaccine or other cancer vaccine.

In some embodiments, the disease is an infectious disease. In someembodiments, the infectious disease is a chronic infectious disease. Insome embodiments, the infectious disease is a chronic viral infection, achronic bacterial infection, or a chronic parasitic infection. In someembodiments, the chronic viral infection is HIV, hepatitis B, hepatitisC. In some embodiments, the chronic bacterial infection is tuberculosis,lyme disease, meningitis, Q fever, ehrlichiosis, bacterial vaginosis,pelvic inflammatory disease, rheumatic fever. In some embodiments, thechronic parasitic infection is malaria, Chagas disease, or isosporiasis.

In some embodiments, the bacterial infection is a severe bacterialinfection of the intestine, wherein the one or more agents areadministered in an amount sufficient to enhance MAIT cell function.

In another aspect, the invention provides a method of treating a chronicinflammatory diseases comprising administering to a subject in needthereof one or more agents in an amount sufficient to either increaseexpression of KLRB1 and/or increase expression of one or more genesencoding CD161 ligands, or to activate or stimulate cell signalingthrough CD161.

In some embodiments, the one or more agents is an agonistic antibody ofCD161. In some embodiments, the one or more CD161 ligands comprisesCLEC2D.

In some embodiments, the chronic inflammatory disease comprises anautoimmune disease.

In another aspect, the invention provides an isolated T cell modified tocomprise decreased expression or activity of, or modified to comprise anagent capable of decreased or increased expression or KLRB1 or activityof CD161.

In some embodiments, the T cell is a CD8+ T cell. In some embodiments,the T cell is a CD4+ T cell.

In some embodiments, the T cell is obtained from peripheral bloodmononuclear cells (PBMCs).

In some embodiments, the T cell is an autologous T cell from a subjectin need of treatment. In some embodiments, the T cell is a TIL obtainedfrom a subject in need of treatment.

In some embodiments, the T cell comprises a chimeric antigen receptor(CAR) or an exogenous T-cell receptor (TCR). In some embodiments, theexogenous TCR is clonally expanded in a tumor. In some embodiments, theCAR or TCR is specific for a tumor antigen. In some embodiments, thetumor antigen is EGFRvIII, Her2, other tumore surface antigen.

In some embodiments, the tumor antigen may be selected from the groupconsisting of: B cell maturation antigen (BCMA); PSA (prostate-specificantigen); prostate-specific membrane antigen (PSMA); PSCA (Prostate stemcell antigen); Tyrosine-protein kinase transmembrane receptor ROR1;fibroblast activation protein (FAP); Tumor-associated glycoprotein 72(TAG72); Carcinoembryonic antigen (CEA); Epithelial cell adhesionmolecule (EPCAM); Mesothelin; Human Epidermal growth factor Receptor 2(ERBB2 (Her2/neu)); Prostase; Prostatic acid phosphatase (PAP);elongation factor 2 mutant (ELF2M); Insulin-like growth factor 1receptor (IGF-1R); gplOO; BCR-ABL (breakpoint cluster region-Abelson);tyrosinase; New York esophageal squamous cell carcinoma 1 (NY-ESO-1);K-light chain, LAGE (L antigen); MAGE (melanoma antigen);Melanoma-associated antigen 1 (MAGE-A1); MAGE A3; MAGE A6; legumain;Human papillomavirus (HPV) E6; HPV E7; prostein; survivin; PCTA1(Galectin 8); Melan-A/MART-1; Ras mutant; TRP-1 (tyrosinase relatedprotein 1, or gp75); Tyrosinase-related Protein 2 (TRP2); TRP-2/INT2(TRP-2/intron 2); RAGE (renal antigen); receptor for advanced glycationend products 1 (RAGE1); Renal ubiquitous 1, 2 (RU1, RU2); intestinalcarboxyl esterase (iCE); Heat shock protein 70-2 (HSP70-2) mutant;thyroid stimulating hormone receptor (TSHR); CD123; CD171; CD19; CD20;CD22; CD26; CD30; CD33; CD44v7/8 (cluster of differentiation 44, exons7/8); CD53; CD92; CD100; CD148; CD150; CD200; CD261; CD262; CD362; CS-1(CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-likemolecule-1 (CLL-1); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (Tn Ag);Fms-Like Tyrosine Kinase 3 (FLT3); CD38; CD138; CD44v6; B7H3 (CD276);KIT (CD 117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2);Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen(PSCA); Protease Serine 21 (PRSS21); vascular endothelial growth factorreceptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growthfactor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4(SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16 (MUC16);epidermal growth factor receptor (EGFR); epidermal growth factorreceptor variant III (EGFRvIII); neural cell adhesion molecule (NCAM);carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit,Beta Type, 9 (LMP2); ephrin type-A receptor 2 (EphA2); Ephrin B2;Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; high molecularweight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside(OAcGD2); Folate receptor alpha; Folate receptor beta; tumor endothelialmarker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R);claudin 6 (CLDN6); G protein-coupled receptor class C group 5, member D(GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a;anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1(PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH);mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2);Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3(ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2(OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumorprotein (WT1); ETS translocation-variant gene 6, located on chromosome12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A(XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); CT(cancer/testis (antigen)); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53;p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin Bi; Cyclin Di; v-myc avian myelocytomatosisviral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog FamilyMember C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor(Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma AntigenRecognized By T Cells-1 or 3 (SART1, SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint-1, -2, -3 or -4 (SSX1, SSX2, SSX3, SSX4);CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1(LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300molecule-like family member f (CD300LF); C-type lectin domain family 12member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyteantigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); mousedouble minute 2 homolog (MDM2); livin; alphafetoprotein (AFP);transmembrane activator and CAML Interactor (TACI); B-cell activatingfactor receptor (BAFF-R); V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog (KRAS); immunoglobulin lambda-like polypeptide 1 (IGLL1); 707-AP(707 alanine proline); ART-4 (adenocarcinoma antigen recognized by T4cells); BAGE (B antigen; b-catenin/m, b-catenin/mutated); CAMEL(CTL-recognized antigen on melanoma); CAPi (carcinoembryonic antigenpeptide 1); CASP-8 (caspase-8); CDC27m (cell-division cycle 27 mutated);CDK4/m (cycline-dependent kinase 4 mutated); Cyp-B (cyclophilin B); DAM(differentiation antigen melanoma); EGP-2 (epithelial glycoprotein 2);EGP-40 (epithelial glycoprotein 40); Erbb2, 3, 4 (erythroblasticleukemia viral oncogene homolog-2, -3, 4); FBP (folate binding protein);fAchR (Fetal acetylcholine receptor); G250 (glycoprotein 250); GAGE (Gantigen); GnT-V (N-acetylglucosaminyltransferase V); HAGE (helicoseantigen); ULA-A (human leukocyte antigen-A); HST2 (human signet ringtumor 2); KIAA0205; KDR (kinase insert domain receptor); LDLR/FUT (lowdensity lipid receptor/GDP L-fucose: b-D-galactosidase 2-a-Lfucosyltransferase); L1CAM (L1 cell adhesion molecule); MC1R(melanocortin 1 receptor); Myosin/m (myosin mutated); MUM-1, -2, -3(melanoma ubiquitous mutated 1, 2, 3); NA88-A (NA cDNA clone of patientM88); KG2D (Natural killer group 2, member D) ligands; oncofetal antigen(h5T4); p190 minor ber-abl (protein of 190KD ber-abl); Pml/RARa(promyelocytic leukaemia/retinoic acid receptor a); PRAME(preferentially expressed antigen of melanoma); SAGE (sarcoma antigen);TEL/AML1 (translocation Ets-family leukemia/acute myeloid leukemia 1);TPI/m (triosephosphate isomerase mutated); CD70; and any combinationthereof.

In some embodiments, the T cell may be further modified to comprisedecreased expression or activity of, or modified to comprise an agentcapable of decreasing expression or activity of a gene or polypeptideselected from the group consisting of TOB1, RGS1, TARP, NKG7, CCL4 andany combination thereof.

In some embodiments, the T cell may be further modified to comprisedecreased expression or activity of the T cell receptor alpha constantchain locus (TRAC).

In some embodiments, the T cell may be activated.

In some embodiments, the T cell may be modified using a CRISPR systemcomprising guide sequences specific to the target. In some embodiments,the CRISPR system may comprise Cas9, Cas12, Cas13, or Cas14.

In another aspect, the invention provides a population of T cellscomprising the T cells described herein.

In another aspect, the invention provides a pharmaceutical compositioncomprising the population of T cells described herein.

In another aspect, the invention provides a method of treating cancer ina subject in need thereof comprising administering the pharmaceuticalcomposition as described herein to the subject.

In some embodiments, the population of cells may be administered byinfusion into the cerebral spinal fluid (CSF). In some embodiments, thepopulation of cells may be administered by injection into the cerebralspinal fluid (CSF) through the lateral ventricle.

In some embodiments, the population of cells may be administered in acombination treatment regimen comprising checkpoint blockade therapy. Insome embodiments, the checkpoint blockade therapy comprises anti-PD-1,anti-CTLA4, anti-PDL1, anti-TIM-3 and/or anti-LAG3.

In some embodiments, the cancer expresses CLEC2D. In some embodiments,tumor infiltrating lymphocytes (TILs) in the cancer express KLRB1. Insome embodiments, the cancer is glioblastoma multiforme (GBM).

The administration of the one or more agents may be based on firstdetermining if a sample obtained from the disease compartment of thesubject is characterized by increased expression of KLRB1 as compared toa control and/or increased expression of one or more genes encoding oneor more CD161 ligands as compared to a control.

These and other aspects, objects, features, and advantages of theexample embodiments will become apparent to those having ordinary skillin the art upon consideration of the following detailed description ofillustrated example embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the present inventionwill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of theinvention may be utilized, and the accompanying drawings of which:

An understanding of the features and advantages of the present inventionwill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of theinvention may be utilized, and the accompanying drawings of which:

FIGS. 1A-1B—provides diagrams of mRNA-seq using SMART-seq2 in accordancewith certain example embodiments.

FIG. 2—provides a schematic for electroporation based delivery of RNPcomplex, in accordance with certain example embodiments.

FIGS. 3A-3C—provides a schematic for editing isolated T-cells (FIG. 3A)and results of editing efficiency of KLRB1 (FIGS. 3B and 3C).

FIGS. 4A-4C—(FIG. 4A, 4B) provide schematics of injection sites for CART cells to interrogate GBM-specific negative regulator roles in vitroand in vivo; and (FIG. 4C) image of control injection in mouse brain.

FIG. 5A summarizes GBM allocation processing and FIG. 5B shows data forpatient groups examined.

FIGS. 6A, 6B—graphs illustrating that GBM tumor-infiltrating T cellshave low T cell activation despite only moderate expression of PD-1 byflow cytometry analysis of clinical GBM-specimens obtained from theDFCI/BWH clinic.

FIG. 7—shows results of GBM-infiltrating T cell single cell analyses.

FIGS. 8A, 8B—illustrates the results of single-cell RNAseq analyses of Tcells in GBM. FIG. 8A Shows the identification of major T cellsubpopulations. Cyto refers to cytotoxic signature. FIG. 8B Shows geneexpression analysis results of clonal versus non-clonal T cells based onexpression (Y-axis) and variability (X-axis) among clones. KLRB1 (box)stands out as one of the genes with the highest expression and leastvariability in clonal T cells compared to non-clonal T cells. PDCD1 andCTLA4 are shown with arrows.

FIG. 9—provides expression analysis results of clonal versus non-clonalT cells based on expression of (Y-axis) and variability (X-axis) amongclones. KLRB1 (box) demonstrates high expression and low variability inclonal T cells as compared to non-clonal T cells.

FIG. 10—shows a summary of genes by CD4 clonotypes with single-cellRNAseq analyses.

FIGS. 11A, 11B—show gene expression analysis results of clonal versusnon-clonal (FIG. 11A) CD4+ and (FIG. 11B) CD8+ T cells expressing KLRB1.

FIGS. 12A-12C—shows expression of KLRB1 in different T cell clusters.

FIGS. 13A, 13B—show results of single cell RNA-seq analysis of T cellsin GBM.

FIGS. 14A, 14B—illustrate that KLRB1 is expressed at high levels byeffector CD8 and CD4 T cells, but not by CD4 Tregs.

FIGS. 15A, 15B—show flow cytometry data illustrating high-levelexpression of CD161 by GBM-infiltrating (FIG. 15A) CD8 T cells or (FIG.15B) CD4 T cells in a patient with recurrent GBM. Live, single T-cells(gating: Calcein AM+, Exclusion−, CD45+, CD3+, CD8+ or CD4+) wereanalyzed for the expression of CD161-BV421 and PD1-PE.

FIG. 16—shows a heatmap illustrating the cytotoxic signature by CD8 Tcells and subset of CD4 T cells in GBM of new onset patients.

FIG. 17—shows a heatmap illustrating the cytotoxic signature by CD8 Tcells and subset of CD4 T cells in GBM of recurrent patients.

FIGS. 18A, 18B—show graphs illustrating TraCeR reconstruction of T cellclonotypes across patients. Percent expanded T cells: CD4+ (non-Treg)11%; Treg 22%; CD8+ 25%.

FIGS. 19A-19D—show pie charts illustrating that CD8 T cells that expressKLRB1 and other NK cell markers have diverse T cell receptors and aretherefore not MAIT cells.

FIGS. 20A-20D—show pie charts illustrating that CD4 T cells that expresshigh levels of KLRB1 have diverse T cell receptors and are therefore notNKT cells.

FIGS. 21A, 21B—heatmaps showing examples of (FIG. 21A) E42 CD4 and (FIG.21B) E42 CD8 clonotypes with high KLRB1 expression.

FIGS. 22A, 22B—illustrate KLRB1 expression in clonal versus non-clonal(FIG. 22A) CD4 T cells or (FIG. 22B) CD8 T cells (number of cells inclonotype ≥2).

FIGS. 23A, 23B—show data illustrating that KLRB1 is expressed at ahigher level by T cells in GBM compared to melanoma. In FIG. 23A, geneexpression was averaged across all T cells. In FIG. 23B, gene expressionwas averaged across CD8_CD8NK T cells.

FIGS. 24A-24E—show results of single cell RNA-seq analysis ofglioblastoma (Itay cohort). FIG. 24A shows GBM cell subsets. FIGS. 24Band 24C show CLEC2B and CLEC2D expression in myeloid cells and T cells;and FIGS. 24D and 24E show CLEC2D expression in malignant cells.

FIGS. 25A-25C—FIG. 25A shows a schematic for the method used forisolation of CD161+ T cell population from primary human T cells forfunctional studies. FIGS. 25B and 25C show FACS data obtained usingthese cells.

FIGS. 26A, 26B—graphs showing the efficiency of simultaneous knockout ofTRAC and KLRB1 genes, with FIG. 26A showing the editing efficiency forKLRB1 and FIG. 26B showing the editing efficiency for TRAC.

FIGS. 27A-27C—illustrate expression of NY-ESO-1 TCR in edited T cells.FIG. 27A shows the lentiviral construct used to transduce primary TRACand KLRB1 edited CD161+ human T cells. FIG. 27B shows results forcontrol edited T cells and FIG. 27C shows results for KLRB1 edited Tcells.

FIGS. 28A, 28B—graphs illustrating that inactivation of the KLRB1 geneenhances T cell function following interaction with GBM cells. Data areshown for (FIG. 28A) CD4+ and (FIG. 28B) CD8+ T cells in a 24 hourco-culture. * P<0.05, ** P<0.01, *** P<0.001.

FIGS. 29A, 29B—graphs illustrating that inactivation of the KLRB1 geneenhances T cell function following interaction with GBM cells. Data areshown for (FIG. 29A) CD4+ and (FIG. 29B) CD8+ T cells in a 48 hourco-culture. * P<0.05, *** P<0.001.

FIGS. 30A, 30B—graphs showing that inactivation of the KLRB1 geneenhances cytokine production by T cells. Shown are results for (FIG.30A) IFNγ and (FIG. 30B) IL-2. ** P<0.01, *** P<0.001, **** P<0.0001.

FIGS. 31A, 31B—graphs showing that inactivation of the KLRB1 genegreatly reduces PD-1 expression by T cells. Engineered NYE-ESO-1 TCR+ Tcells (NYE_TCR) that were edited for LacZ or KLRB1 gRNAs, were examinedby flow cytometry at (FIG. 31A) 48h or (FIG. 31B) 72h followingcoculture with U87MG cells that express NY-ESO-1 peptide at theindicated effector to target ratios. ** P<0.01, *** P<0.001, ****P<0.0001.

FIG. 32—flow cytometery data showing that KLRB1 edited human T cellsexhibit increased activation and decreased inhibitory expression of thePD-1 inhibitory receptor in vivo.

FIG. 33—graph shows a survival analysis following transfer of KLRB1edited or control edited human T cells.

FIG. 34—schematic of stereotactic surgical procedures for xenograftmodel of GBM.

For any figure showing a bar histogram, curve, or other data associatedwith a legend, the bars, curve, or other data presented from left toright for each indication correspond directly and in order to the boxesfrom top to bottom, or from left to right, of the legend.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS General Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Definitions of common termsand techniques in molecular biology may be found in Molecular Cloning: ALaboratory Manual, 2^(nd) edition (1989) (Sambrook, Fritsch, andManiatis); Molecular Cloning: A Laboratory Manual, 4^(th) edition (2012)(Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (AcademicPress, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B.D. Hames, and G. R. Taylor eds.): Antibodies, A Laboratory Manual (1988)(Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2^(nd) edition2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney,ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008(ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829);Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology andMolecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed.,John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Janvan Deursen, Transgenic Mouse Methods and Protocols, 2^(nd) edition(2011).

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The term “optional” or “optionally” means that the subsequent describedevent, circumstance or substituent may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The terms “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, are meant to encompass variations of and from thespecified value, such as variations of +/−10% or less, +/−5% or less,+/−1% or less, and +/−0.1% or less of and from the specified value,insofar such variations are appropriate to perform in the disclosedinvention. It is to be understood that the value to which the modifier“about” or “approximately” refers is itself also specifically, andpreferably, disclosed.

As used herein, a “biological sample” may contain whole cells and/orlive cells and/or cell debris. The biological sample may contain (or bederived from) a “bodily fluid”. The present invention encompassesembodiments wherein the bodily fluid is selected from amniotic fluid,aqueous humour, vitreous humour, bile, blood serum, breast milk,cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph,perilymph, exudates, feces, female ejaculate, gastric acid, gastricjuice, lymph, mucus (including nasal drainage and phlegm), pericardialfluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skinoil), semen, sputum, synovial fluid, sweat, tears, urine, vaginalsecretion, vomit and mixtures of one or more thereof. Biological samplesinclude cell cultures, bodily fluids, cell cultures from bodily fluids.Bodily fluids may be obtained from a mammal organism, for example bypuncture, or other collecting or sampling procedures.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

Various embodiments are described hereinafter. It should be noted thatthe specific embodiments are not intended as an exhaustive descriptionor as a limitation to the broader aspects discussed herein. One aspectdescribed in conjunction with a particular embodiment is not necessarilylimited to that embodiment and can be practiced with any otherembodiment(s). Reference throughout this specification to “oneembodiment”, “an embodiment,” “an example embodiment,” means that aparticular feature, structure or characteristic described in connectionwith the embodiment is included in at least one embodiment of thepresent invention. Thus, appearances of the phrases “in one embodiment,”“in an embodiment,” or “an example embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment, but may. Furthermore, the particular features,structures or characteristics may be combined in any suitable manner, aswould be apparent to a person skilled in the art from this disclosure,in one or more embodiments. Furthermore, while some embodimentsdescribed herein include some but not other features included in otherembodiments, combinations of features of different embodiments are meantto be within the scope of the invention. For example, in the appendedclaims, any of the claimed embodiments can be used in any combination.

All publications, published patent documents, and patent applicationscited herein are hereby incorporated by reference to the same extent asthough each individual publication, published patent document, or patentapplication was specifically and individually indicated as beingincorporated by reference.

Overview

Embodiments disclosed herein provide methods and compositions fortreating diseases characterized by increased expression of Killer CellLectin Like Receptor (KLRB1 receptor and also known as CD161, HNKR-P1A,HNKR-P1a, NKR-P1, and NKR) on immune cells, by targeting theCLEC2D-KLRB1 pathway. The function of the KLRB1 receptor in T cells waspreviously not known. KLRB1 is only expressed by small subpopulations ofhuman blood T cells. KLRB1 ligands include C-type Lectin Domain Family 2Member D, (CLEC2D and also known as LLT-1, CLAX, LLT1, OCIL). As shownherein, KLRB1 expression is induced in T cells in a cancermicroenvironment. Inactivation of KLRB1 in said T cells demonstratedhigher levels of tumor control. Accordingly, KLRB1 appears to be animportant regulator of, at least, T cell function. Given this keyregulatory role, embodiments disclosed herein are directed to treatingdiseases characterized by increased expression of KLRB1. Accordingly, inone aspect, embodiments disclosed herein provide agents, compositions,and methods for treating diseases where immune function and/or T cellfunction may need to be increased by inhibiting KLRB1 expression and/orligand binding, such as in the treatment of cancer and infectiousdisease. Likewise, in another aspect, embodiments disclosed hereinprovide agents, compositions and methods for treating diseases whereimmune function and/or T cell function may need to be repressed byincreasing KLRB1 expression and/or ligand binding, such as in chronicinflammatory and autoimmune diseases.

In one embodiment, compositions and method are provided for treatingdisease characterized by increased expression of KLRB1 in immune cellscomprising administering to a subject in need thereof an effectiveamount of one or more agents sufficient to blocking binding of KLRB1 toone or more KLRIB ligands. In certain example embodiments, immune cellsexhibiting increase KLRB1 expression are within the diseasemicroenvironment. As used herein, a “disease microenvironment” refers tothe cellular environment in which diseased cells or tissue exist and mayinclude the surrounding blood vessels, immune cells, fibroblasts,bone-marrow derived inflammatory cells, lymphocytes, signaling moleculesand the extracellular matrix. A disease microenvironment may furthercharacterized by closely related and constant interactions between thediseased cells and immune cells responding to the presence of thedisease cells. For example, in a tumor microenvironment, tumor cells caninfluence the microenvironment by releasing extracellular signals,promoting tumor angiogenesis and inducing peripheral immune tolerance,while the immune cells in the microenvironment can affect the growth andevolution of cancer cells.

In another embodiment, compositions and methods are provided fortreating disease characterized by increased KLRB1 expression in immunecells, comprising administering to a subject in need thereof, one ormore agents in an amount sufficient to block binding of KLRBI1 to one ormore KLRBI1 ligands. In one example, the KLRB1 ligands comprises CLEC2D.

In another embodiment, compositions and methods are provided fortreating diseases characterized by increased KLRBI1 expression in immunecells comprising administering to a subject in need thereof one or moreagents in an amount sufficient to reduce expression of KLRB1 or one ormore KLRB1 ligands. In certain example embodiments, the one or moreKLRB1 ligands comprises CLEC2D.

In another embodiment, compositions and methods are provided fortreating diseases characterized by increased KLRBI1 expression in immunecells comprising administering one or more agents in an amountsufficient to block binding of CLEC2D to a receptor of CLEC2D other thanKLRB1.

In another embodiment, compositions and method for treating chronicinflammatory diseases, including autoimmune diseases, comprisingadministering to a subject in need thereof of one or more agents in anamount sufficient to increase expression of KLRB1 or one or more KLRB1ligands, or increase activation and cell signaling via KLRB1. In certainexample embodiments, the one or more KLRB1 ligands comprises CLEC2D.

In another aspects, embodiments disclosed herein provide isolated Tcells modified to decrease or increase expression of KLRB1. In certainexample embodiments, the isolated T cells are modified to decreaseexpression of KLRB1. In certain other example embodiments, the isolatedT cells are modified to increase expression of KLRB1. Further disclosedare cell populations and therapeutic compositions comprising saidmodified T cells, and methods of use thereof In certain exampleembodiments, a method of treating comprises administering said modifiedT cells, wherein the modified T cells have reduced expression of KLRB1.

Further descriptions of therapeutic agents for use in these and otherembodiments, as well as methods of treatment, diagnosis and screeningare described in further detail below.

Therapeutic Agents

Embodiments disclosed herein comprise agents that target theCLEC2D-KLRB1 inhibitory signaling pathway. In certain exampleembodiments, the agent may inhibit or reduce signaling through KLRB1. Incertain example embodiments, the agent may block binding of KLRB1 to oneor more KLRB1 ligands. In certain other example embodiments, the agentmay reduce expression of KLRB1. In certain other example embodiments,the agent may reduce expression of one or more KLRB1 ligands. KLRB1ligands include, for example, CLEC2D. In certain example embodiment, theagent may blocking binding of CLEC2D to receptors other than KLRB1.Embodiments disclosed herein may use more than one agent in combinationand each agent may have the same or a different inhibitor effect.

In certain other example embodiments, the agent may increase expressionof KLRB1 or signaling through KLRB1. For example, in the context ofdisease where reduction or inhibition of the immune response is desired,for example in chronic inflammatory conditions.

Protein Binding Agent

In certain embodiments, an “agent” can refer to a protein-binding agentthat permits modulation of activity of proteins or disrupts interactionsof proteins and other biomolecules, such as but not limited todisrupting protein-protein interaction, ligand-receptor interaction, orprotein-nucleic acid interaction. The terms “fragment,” “derivative” and“analog” when referring to polypeptides as used herein refers topolypeptides which either retain substantially the same biologicalfunction or activity as such polypeptides. An analog includes aproprotein which can be activated by cleavage of the proprotein portionto produce an active mature polypeptide. Such agents include, but arenot limited to, antibodies (“antibodies” includes antigen-bindingportions of antibodies such as epitope- or antigen-binding peptides,paratopes, functional CDRs; recombinant antibodies; chimeric antibodies;humanized antibodies; nanobodies; tribodies; midibodies; orantigen-binding derivatives, analogs, variants, portions, or fragmentsthereof), protein-binding agents, nucleic acid molecules, smallmolecules, recombinant protein, peptides, aptamers, avimers andprotein-binding derivatives, portions or fragments thereof.

In certain embodiments, the agent is capable of inhibiting or blockingthe interaction of KLRB1 with its ligand. Such agents may also bereferred to as KLRB1 inhibitors or antagonists and can inhibit eitherthe expression and/or the ability of KLRB1 to bind with its ligand. Insome embodiments, the KLRB1 ligand is CLEC2D. In some embodiments, KLRB1expression is inhibited, e.g., by a DNA targeting agent (e.g., CRISPRsystem, TALE, Zinc finger protein) or an RNA targeting agent (e.g.,inhibitory nucleic acid molecules). In some embodiments, KLRB1 activityis inhibited. Such inhibition includes, e.g., reducing the expression ofits ligand, CLEC2D, or by blocking the interaction of KLRB1 with CLEC2D.In certain embodiments, the antagonist is an antibody or fragmentthereof In certain embodiments, the antibody is specific for KLRB1 orCLEC2D. In some embodiments, the agent is a soluble KLRB1 protein orfragment thereof used to inhibit binding of KLRB1 ligans to KLRB1 cellson the cell surface. In certain other example embodiments, the agent isa soluble KLRB1 ligand, or KLRB1 binding fragment thereof, for use inbinding and activating or stimulating signaling via KLRB1.

Antibodies

In some embodiments, the agent may be an antibody or fragment thereofThe term “antibody” (e.g., anti-KLRB1 or anti-CLEC2D antibody) is usedinterchangeably with the term “immunoglobulin” herein, and includesintact antibodies, fragments of antibodies, e.g., Fab, F(ab′)2fragments, and intact antibodies and fragments that have been mutatedeither in their constant and/or variable region (e.g., mutations toproduce chimeric, partially humanized, or fully humanized antibodies, aswell as to produce antibodies with a desired trait, e.g., enhancedbinding and/or reduced FcR binding). The term “fragment” refers to apart or portion of an antibody or antibody chain comprising fewer aminoacid residues than an intact or complete antibody or antibody chain.Fragments can be obtained via chemical or enzymatic treatment of anintact or complete antibody or antibody chain. Fragments can also beobtained by recombinant means. Exemplary fragments include Fab, Fab′,F(ab′)2, Fabc, Fd, dAb, VHH and scFv and/or Fv fragments.

In some embodiments, the antibody is a humanized or chimeric antibody.“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, FR residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

As used herein, a “blocking” antibody or an antibody “antagonist” is onewhich inhibits or reduces biological activity of the antigen(s) itbinds. For example, an antagonist antibody may bind KLRB1 or CLEC2D andinhibit their ability to interact. In certain embodiments, the blockingantibodies or antagonist antibodies or portions thereof described hereincompletely inhibit the biological activity of the antigen(s). As usedherein, an “agonist” antibody refers to an antibody that binds to KLRB1and stimulates or activates signaling through KLRB1.

Antibodies may act as agonists or antagonists of the recognizedpolypeptides. For example, the present invention includes antibodieswhich disrupt receptor/ligand interactions either partially or fully.The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orof one of its down-stream substrates by immunoprecipitation followed bywestern blot analysis. In specific embodiments, antibodies are providedthat inhibit ligand activity or receptor activity by at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex. Likewise, encompassed by theinvention are neutralizing antibodies which bind the ligand and preventbinding of the ligand to the receptor, as well as antibodies which bindthe ligand, thereby preventing receptor activation, but do not preventthe ligand from binding the receptor. Further included in the inventionare antibodies which activate the receptor. These antibodies may act asreceptor agonists, i.e., potentiate or activate either all or a subsetof the biological activities of the ligand-mediated receptor activation,for example, by inducing dimerization of the receptor. The antibodiesmay be specified as agonists, antagonists or inverse agonists forbiological activities comprising the specific biological activities ofthe peptides disclosed herein. The antibody agonists and antagonists canbe made using methods known in the art. See, e.g., PCT publication WO96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988(1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al.,J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res.58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179(1998); Prat et al., J. Cell. Sci. III (Pt2):237-247 (1998); Pitard etal., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al.,Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem.272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995);Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(1):14-20 (1996).

The antibodies as defined for the present invention include derivativesthat are modified, i.e., by the covalent attachment of any type ofmolecule to the antibody such that covalent attachment does not preventthe antibody from generating an anti-idiotypic response. For example,but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited tospecific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

In certain example embodiments, the antibody may bind an epitope withinamino acids 67-225. All amino acids positioned described herein are into reference of the amino acid sequence described at UniProt Ref #Q12918 KLRB1_HUMAN. Corresponding positions in orthologues or otherexpressed variants are likewise considered. In certain exampleembodiments, the antibody may bind an epitope located with the C-typelectin domain. In certain other example embodiments, the antibody maybind to an epitope between amino acids 101-211.

Example KLRB1 antibodies may include, for example, antibody Cat #:MAB7448 (R&D systems), antibodies AM20021PU-N and AM20021RP-N(Origene),antibodies Cat #: MA1-81379, Cat #: PA5-50375, Cat #: MA5-17537, Cat #:PA5-51096, Cat #: 53-1619-42 (ThermoFisher Scientific), antibodiesNBP2-14845, MAB7448, NBP2-14844, NBP1-88130, NB100-65298, NB100-65297,H00003820-MO1J, and H00003820-MO1 (Novus Biologicals, Littleton, Colo.),antibody PAA986Hu01 (Cloud-Clone Corp.), antibodies GTX75449, GTX34026,GTX15830, and GTX42370 (GeneTex, Irvine, Calif.), CD161 Antibody(B199.2): sc-58963, and CD161 Antibody (HP-3G10): sc-69891 (Santa CruzBiotechnology), etc. Moreover, multiple siRNA, shRNA, CRISPR constructsfor reducing KLRB1 expression can be found in the commercial productlists of the above-referenced companies, such as shRNA product #TR311864, siRNA product # SR302582, SR405669, and SR502504, and CRISPRproduct # KN216459 from Origene Technologies (Rockville, Md.), CRISPRproduct K6654508 (Abm), si/shRNA products sc-42935 and sc-148601, andCRISPR products sc-405895 and sc-405895-KO-2 (Santa Cruz Biotechnology).It is to be noted that the term can further be used to refer to anycombination of features described herein regarding KLRB1 molecules. Forexample, any combination of sequence composition, percentage identify,sequence length, domain structure, functional activity, etc. can be usedto describe an KLRB1 molecule of the present invention.

Example Anti-CLEC2D antibodies may include Cat #: AF3480 and AF3376 (R&Dsystems), antibodies AP23056PU-N, TA336182, and TA349280 (Origene),antibodies Cat #: PA5-53617, Cat #: PA5-42581, Cat #: PA5-47496, and Cat#: MA1-41278 (ThermoFisher Scientific), antibodies H00029121-MO1,NB100-56553, NBP1-84455, H00029121-D01P, NBP1-52380, H00029121-B01P,H00029121-M03, FAB3480A, and FAB3480P (Novus Biologicals, Littleton,Colo.), etc. Moreover, multiple siRNA, shRNA, CRISPR constructs forreducing CLEC2D expression can be found in the commercial product listsof the above-referenced companies, such as shRNA products # TR313860,TF313860, TL313860, and TG313860, siRNA products #SR309218, SR405384,and SR504280, and CRISPR product # KN213794 from Origene Technologies(Rockville, Md.), CRISPR product K6994408 (Abm), si/shRNA productssc-95672 and sc-72014, and CRISPR products sc-412035 (Santa CruzBiotechnology), shRNA product Cat # SH811924, SH884895, SH889166,SH818531, and SH820950 (Vigene Biosciences), etc. It is to be noted thatthe term can further be used to refer to any combination of featuresdescribed herein regarding CLEC2D molecules. For example, anycombination of sequence composition, percentage identify, sequencelength, domain structure, functional activity, etc. can be used todescribe an CLEC2D molecule of the present invention.

Programmable Nucleic-Acid Modifying Agents

An “agent” as used herein, may also refer to an agent that inhibitsexpression of a gene, such as but not limited to a DNA targeting agent(e.g., CRISPR system, TALE, Zinc finger protein) or RNA targeting agent(e.g., inhibitory nucleic acid molecules such as RNAi, miRNA, ribozyme).Programmable nucleic acid-modifying agents in the context of the presentinvention may be used to modify endogenous cell DNA or RNA sequences,including DNA and/or RNA sequences encoding the target genes and targetgene products disclosed herein including KLRB1 and CLEC2D. In certainexample embodiments, the programmable nucleic acid-modifying agents maybe used to edit a target sequence to restore native or wild-typefunctionality. In certain other embodiments, the programmablenucleic-acid modifying agents may be used to insert a new gene or geneproduct to modify the phenotype of target cells. In certain otherexample embodiments, the programmable nucleic-acid modifying agents maybe used to delete or otherwise silence the expression of a target geneor gene product. Programmable nucleic-acid modifying agents may used inboth in vivo an ex vivo applications disclosed herein.

1. CRISPR/Cas Systems

In general, a CRISPR-Cas or CRISPR system as used herein and indocuments, such as WO 2014/093622 (PCT/US2013/074667), referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, a tracr(trans-activating CRISPR) sequence (e.g. tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNAand transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). See, e.g, Shmakov et al. (2015) “Discovery and FunctionalCharacterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell,DOI: dx.doi.org/10.1016/j.molcel.2015.10.008.

In certain embodiments, a protospacer adjacent motif (PAM) or PAM-likemotif directs binding of the effector protein complex as disclosedherein to the target locus of interest. In some embodiments, the PAM maybe a 5′ PAM (i.e., located upstream of the 5′ end of the protospacer).In other embodiments, the PAM may be a 3′ PAM (i.e., located downstreamof the 5′ end of the protospacer). The term “PAM” may be usedinterchangeably with the term “PFS” or “protospacer flanking site” or“protospacer flanking sequence”.

In a preferred embodiment, the CRISPR effector protein may recognize a3′ PAM. In certain embodiments, the CRISPR effector protein mayrecognize a 3′ PAM which is 5′H, wherein His A, C or U.

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise RNA polynucleotides. The term “target RNA” refersto a RNA polynucleotide being or comprising the target sequence. Inother words, the target RNA may be a RNA polynucleotide or a part of aRNA polynucleotide to which a part of the gRNA, i.e. the guide sequence,is designed to have complementarity and to which the effector functionmediated by the complex comprising CRISPR effector protein and a gRNA isto be directed. In some embodiments, a target sequence is located in thenucleus or cytoplasm of a cell.

In certain example embodiments, the CRISPR effector protein may bedelivered using a nucleic acid molecule encoding the CRISPR effectorprotein. The nucleic acid molecule encoding a CRISPR effector protein,may advantageously be a codon optimized CRISPR effector protein. Anexample of a codon optimized sequence, is in this instance a sequenceoptimized for expression in eukaryote, e.g., humans (i.e. beingoptimized for expression in humans), or for another eukaryote, animal ormammal as herein discussed; see, e.g., SaCas9 human codon optimizedsequence in WO 2014/093622 (PCT/US2013/074667). Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs is known. In some embodiments, anenzyme coding sequence encoding a CRISPR effector protein is a codonoptimized for expression in particular cells, such as eukaryotic cells.The eukaryotic cells may be those of or derived from a particularorganism, such as a plant or a mammal, including but not limited tohuman, or non-human eukaryote or animal or mammal as herein discussed,e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal orprimate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. In general,codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g. about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at kazusa.orjp/codon/ and these tables can beadapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codonoptimizing a particular sequence for expression in a particular hostcell are also available, such as Gene Forge (Aptagen; Jacobus, PA), arealso available. In some embodiments, one or more codons (e.g. 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encodinga Cas correspond to the most frequently used codon for a particularamino acid.

In certain embodiments, the methods as described herein may compriseproviding a Cas transgenic cell in which one or more nucleic acidsencoding one or more guide RNAs are provided or introduced operablyconnected in the cell with a regulatory element comprising a promoter ofone or more gene of interest. As used herein, the term “Cas transgeniccell” refers to a cell, such as a eukaryotic cell, in which a Cas genehas been genomically integrated. The nature, type, or origin of the cellare not particularly limiting according to the present invention. Alsothe way the Cas transgene is introduced in the cell may vary and can beany method as is known in the art. In certain embodiments, the Castransgenic cell is obtained by introducing the Cas transgene in anisolated cell. In certain other embodiments, the Cas transgenic cell isobtained by isolating cells from a Cas transgenic organism. By means ofexample, and without limitation, the Cas transgenic cell as referred toherein may be derived from a Cas transgenic eukaryote, such as a Casknock-in eukaryote. Reference is made to WO 2014/093622(PCT/US13/74667), incorporated herein by reference. Methods of US PatentPublication Nos. 20120017290 and 20110265198 assigned to SangamoBioSciences, Inc. directed to targeting the Rosa locus may be modifiedto utilize the CRISPR Cas system of the present invention. Methods of USPatent Publication No. 20130236946 assigned to Cellectis directed totargeting the Rosa locus may also be modified to utilize the CRISPR Cassystem of the present invention. By means of further example referenceis made to Platt et. al. (Cell; 159(2):440-455 (2014)), describing aCas9 knock-in mouse, which is incorporated herein by reference. The Castransgene can further comprise a Lox-Stop-polyA-Lox(LSL) cassettethereby rendering Cas expression inducible by Cre recombinase.Alternatively, the Cas transgenic cell may be obtained by introducingthe Cas transgene in an isolated cell. Delivery systems for transgenesare well known in the art. By means of example, the Cas transgene may bedelivered in for instance eukaryotic cell by means of vector (e.g., AAV,adenovirus, lentivirus) and/or particle and/or nanoparticle delivery, asalso described herein elsewhere.

It will be understood by the skilled person that the cell, such as theCas transgenic cell, as referred to herein may comprise further genomicalterations besides having an integrated Cas gene or the mutationsarising from the sequence specific action of Cas when complexed with RNAcapable of guiding Cas to a target locus.

In certain aspects the invention involves vectors, e.g. for deliveringor introducing in a cell Cas and/or RNA capable of guiding Cas to atarget locus (i.e. guide RNA), but also for propagating these components(e.g. in prokaryotic cells). A used herein, a “vector” is a tool thatallows or facilitates the transfer of an entity from one environment toanother. It is a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. Ingeneral, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorsinclude, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses (AAVs)). Viral vectors also includepolynucleotides carried by a virus for transfection into a host cell.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g. bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively-linked. Such vectors are referred to herein as “expressionvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety. Thus, the embodiments disclosed herein mayalso comprise transgenic cells comprising the CRISPR effector system. Incertain example embodiments, the transgenic cell may function as anindividual discrete volume. In other words, samples comprising a maskingconstruct may be delivered to a cell, for example in a suitable deliveryvesicle and if the target is present in the delivery vesicle the CRISPReffector is activated and a detectable signal generated.

The vector(s) can include the regulatory element(s), e.g., promoter(s).The vector(s) can comprise Cas encoding sequences, and/or a single, butpossibly also can comprise at least 3 or 8 or 16 or 32 or 48 or 50 guideRNA(s) (e.g., sgRNAs) encoding sequences, such as 1-2, 1-3, 1-4 1-5,3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s)(e.g., sgRNAs). In a single vector there can be a promoter for each RNA(e.g., sgRNA), advantageously when there are up to about 16 RNA(s); and,when a single vector provides for more than 16 RNA(s), one or morepromoter(s) can drive expression of more than one of the RNA(s), e.g.,when there are 32 RNA(s), each promoter can drive expression of twoRNA(s), and when there are 48 RNA(s), each promoter can drive expressionof three RNA(s). By simple arithmetic and well established cloningprotocols and the teachings in this disclosure one skilled in the artcan readily practice the invention as to the RNA(s) for a suitableexemplary vector such as AAV, and a suitable promoter such as the U6promoter. For example, the packaging limit of AAV is ˜4.7 kb. The lengthof a single U6-gRNA (plus restriction sites for cloning) is 361 bp.Therefore, the skilled person can readily fit about 12-16, e.g., 13U6-gRNA cassettes in a single vector. This can be assembled by anysuitable means, such as a golden gate strategy used for TALE assembly(genome-engineering.org/taleffectors/). The skilled person can also usea tandem guide strategy to increase the number of U6-gRNAs byapproximately 1.5 times, e.g., to increase from 12-16, e.g., 13 toapproximately 18-24, e.g., about 19 U6-gRNAs. Therefore, one skilled inthe art can readily reach approximately 18-24, e.g., about 19promoter-RNAs, e.g., U6-gRNAs in a single vector, e.g., an AAV vector. Afurther means for increasing the number of promoters and RNAs in avector is to use a single promoter (e.g., U6) to express an array ofRNAs separated by cleavable sequences. And an even further means forincreasing the number of promoter-RNAs in a vector, is to express anarray of promoter-RNAs separated by cleavable sequences in the intron ofa coding sequence or gene; and, in this instance it is advantageous touse a polymerase II promoter, which can have increased expression andenable the transcription of long RNA in a tissue specific manner. (see,e.g., nar.oxfordjournals.org/content/34/7/e53.short andnature.com/mt/journal/v16/n9/abs/mt2008144a.html). In an advantageousembodiment, AAV may package U6 tandem gRNA targeting up to about 50genes. Accordingly, from the knowledge in the art and the teachings inthis disclosure the skilled person can readily make and use vector(s),e.g., a single vector, expressing multiple RNAs or guides under thecontrol or operatively or functionally linked to one or morepromoters-especially as to the numbers of RNAs or guides discussedherein, without any undue experimentation.

The guide RNA(s) encoding sequences and/or Cas encoding sequences, canbe functionally or operatively linked to regulatory element(s) and hencethe regulatory element(s) drive expression. The promoter(s) can beconstitutive promoter(s) and/or conditional promoter(s) and/or induciblepromoter(s) and/or tissue specific promoter(s). The promoter can beselected from the group consisting of RNA polymerases, pol I, pol II,pol III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter,the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolatereductase promoter, the β-actin promoter, the phosphoglycerol kinase(PGK) promoter, and the EF1α promoter. An advantageous promoter is thepromoter is U6.

Additional effectors for use according to the invention can beidentified by their proximity to cas1 genes, for example, though notlimited to, within the region 20 kb from the start of the cas1 gene and20 kb from the end of the cas1 gene. In certain embodiments, theeffector protein comprises at least one HEPN domain and at least 500amino acids, and wherein the C2c2 effector protein is naturally presentin a prokaryotic genome within 20 kb upstream or downstream of a Casgene or a CRISPR array. Non-limiting examples of Cas proteins includeCas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also knownas Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2,Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6,Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15,Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereofIn certain example embodiments, the C2c2 effector protein is naturallypresent in a prokaryotic genome within 20 kb upstream or downstream of aCas 1 gene. The terms “orthologue” (also referred to as “ortholog”herein) and “homologue” (also referred to as “homolog” herein) are wellknown in the art. By means of further guidance, a “homologue” of aprotein as used herein is a protein of the same species which performsthe same or a similar function as the protein it is a homologue of.Homologous proteins may but need not be structurally related, or areonly partially structurally related. An “orthologue” of a protein asused herein is a protein of a different species which performs the sameor a similar function as the protein it is an orthologue of Orthologousproteins may but need not be structurally related, or are only partiallystructurally related.

a) DNA repair and NHEJ

In certain embodiments, nuclease-induced non-homologous end-joining(NHEJ) can be used to target gene-specific knockouts. Nuclease-inducedNHEJ can also be used to remove (e.g., delete) sequence in a gene ofinterest. Generally, NHEJ repairs a double-strand break in the DNA byjoining together the two ends; however, generally, the original sequenceis restored only if two compatible ends, exactly as they were formed bythe double-strand break, are perfectly ligated. The DNA ends of thedouble-strand break are frequently the subject of enzymatic processing,resulting in the addition or removal of nucleotides, at one or bothstrands, prior to rejoining of the ends. This results in the presence ofinsertion and/or deletion (indel) mutations in the DNA sequence at thesite of the NHEJ repair. Two-thirds of these mutations typically alterthe reading frame and, therefore, produce a non-functional protein.Additionally, mutations that maintain the reading frame, but whichinsert or delete a significant amount of sequence, can destroyfunctionality of the protein. This is locus dependent as mutations incritical functional domains are likely less tolerable than mutations innon-critical regions of the protein. The indel mutations generated byNHEJ are unpredictable in nature; however, at a given break site certainindel sequences are favored and are over represented in the population,likely due to small regions of microhomology. The lengths of deletionscan vary widely; most commonly in the 1-50 bp range, but they can easilybe greater than 50 bp, e.g., they can easily reach greater than about100-200 bp. Insertions tend to be shorter and often include shortduplications of the sequence immediately surrounding the break site.However, it is possible to obtain large insertions, and in these cases,the inserted sequence has often been traced to other regions of thegenome or to plasmid DNA present in the cells.

Because NHEJ is a mutagenic process, it may also be used to delete smallsequence motifs as long as the generation of a specific final sequenceis not required. If a double-strand break is targeted near to a shorttarget sequence, the deletion mutations caused by the NHEJ repair oftenspan, and therefore remove, the unwanted nucleotides. For the deletionof larger DNA segments, introducing two double-strand breaks, one oneach side of the sequence, can result in NHEJ between the ends withremoval of the entire intervening sequence. Both of these approaches canbe used to delete specific DNA sequences; however, the error-pronenature of NHEJ may still produce indel mutations at the site of repair.

Both double strand cleaving by the CRISPR/Cas system can be used in themethods and compositions described herein to generate NHEJ-mediatedindels. NHEJ-mediated indels targeted to the gene, e.g., a codingregion, e.g., an early coding region of a gene of interest can be usedto knockout (i.e., eliminate expression of) a gene of interest. Forexample, early coding region of a gene of interest includes sequenceimmediately following a transcription start site, within a first exon ofthe coding sequence, or within 500 bp of the transcription start site(e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp).

In an embodiment, in which the CRISPR/Cas system generates a doublestrand break for the purpose of inducing NHEJ-mediated indels, a guideRNA may be configured to position one double-strand break in closeproximity to a nucleotide of the target position. In an embodiment, thecleavage site may be between 0-500 bp away from the target position(e.g., less than 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9,8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position).

In an embodiment, in which two guide RNAs complexing with CRISPR/Cassystem nickases induce two single strand breaks for the purpose ofinducing NHEJ-mediated indels, two guide RNAs may be configured toposition two single-strand breaks to provide for NHEJ repair anucleotide of the target position.

b) dCas and Functional Effectors

Unlike CRISPR-Cas-mediated gene knockout, which permanently eliminatesexpression by mutating the gene at the DNA level, CRISPR-Cas knockdownallows for temporary reduction of gene expression through the use ofartificial transcription factors. Mutating key residues in cleavagedomains of the Cas protein results in the generation of a catalyticallyinactive Cas protein. A catalytically inactive Cas protein complexeswith a guide RNA and localizes to the DNA sequence specified by thatguide RNA's targeting domain, however, it does not cleave the targetDNA. Fusion of the inactive Cas protein to an effector domain alsoreferred to herein as a functional domain, e.g., a transcriptionrepression domain, enables recruitment of the effector to any DNA sitespecified by the guide RNA.

In general, the positioning of the one or more functional domain on theinactivated CRISPR/Cas protein is one which allows for correct spatialorientation for the functional domain to affect the target with theattributed functional effect. For example, if the functional domain is atranscription activator (e.g., VP64 or p65), the transcription activatoris placed in a spatial orientation which allows it to affect thetranscription of the target. Likewise, a transcription repressor will beadvantageously positioned to affect the transcription of the target, anda nuclease (e.g., Fok1) will be advantageously positioned to cleave orpartially cleave the target. This may include positions other than theN-/C-terminus of the CRISPR protein.

In certain embodiments, Cas protein may be fused to a transcriptionalrepression domain and recruited to the promoter region of a gene.Especially for gene repression, it is contemplated herein that blockingthe binding site of an endogenous transcription factor would aid indownregulating gene expression.

In an embodiment, a guide RNA molecule can be targeted to a knowntranscription response elements (e.g., promoters, enhancers, etc.), aknown upstream activating sequences, and/or sequences of unknown orknown function that are suspected of being able to control expression ofthe target DNA. Idem: adapt to refer to regions with the motifs ofinterest

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does. For example, a protein or microRNA codingsequence may be inactivated such that the protein is not produced.

In certain example embodiments, the functional domain may be anucleotide deaminase. The nucleotide deaminase may be a cytidinedeaminase or an adenosine deaminase. In certain example embodiments, thedCas may be a dCas9, a dCas12, a dCas13, or a dCas14. In certain exampleembodiments, the dCas9-nucleotide deaminase may be used to change one ormore post-translation modification sites on KLRB1. In certain exampleembodiments, the post-translation modification site may be a disulfidebond located at amino acid 94, 105, 122, 189, and 202. In anotherexample embodiment, the post-translation modification site may be aglycosylation site at amino acid 157.

c) Guide Molecules

As used herein, the term “guide sequence” and “guide molecule” in thecontext of a CRISPR-Cas system, comprises any polynucleotide sequencehaving sufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a nucleic acid-targeting complex to thetarget nucleic acid sequence. The guide sequences made using the methodsdisclosed herein may be a full-length guide sequence, a truncated guidesequence, a full-length sgRNA sequence, a truncated sgRNA sequence, oran E+F sgRNA sequence. In some embodiments, the degree ofcomplementarity of the guide sequence to a given target sequence, whenoptimally aligned using a suitable alignment algorithm, is about or morethan about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Incertain example embodiments, the guide molecule comprises a guidesequence that may be designed to have at least one mismatch with thetarget sequence, such that a RNA duplex formed between the guidesequence and the target sequence. Accordingly, the degree ofcomplementarity is preferably less than 99%. For instance, where theguide sequence consists of 24 nucleotides, the degree of complementarityis more particularly about 96% or less. In particular embodiments, theguide sequence is designed to have a stretch of two or more adjacentmismatching nucleotides, such that the degree of complementarity overthe entire guide sequence is further reduced. For instance, where theguide sequence consists of 24 nucleotides, the degree of complementarityis more particularly about 96% or less, more particularly, about 92% orless, more particularly about 88% or less, more particularly about 84%or less, more particularly about 80% or less, more particularly about76% or less, more particularly about 72% or less, depending on whetherthe stretch of two or more mismatching nucleotides encompasses 2, 3, 4,5, 6 or 7 nucleotides, etc. In some embodiments, aside from the stretchof one or more mismatching nucleotides, the degree of complementarity,when optimally aligned using a suitable alignment algorithm, is about ormore than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.Optimal alignment may be determined with the use of any suitablealgorithm for aligning sequences, non-limiting example of which includethe Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g., the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies;available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). The ability of a guide sequence (within a nucleicacid-targeting guide RNA) to direct sequence-specific binding of anucleic acid-targeting complex to a target nucleic acid sequence may beassessed by any suitable assay. For example, the components of a nucleicacid-targeting CRISPR system sufficient to form a nucleic acid-targetingcomplex, including the guide sequence to be tested, may be provided to ahost cell having the corresponding target nucleic acid sequence, such asby transfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget nucleic acid sequence (or a sequence in the vicinity thereof) maybe evaluated in a test tube by providing the target nucleic acidsequence, components of a nucleic acid-targeting complex, including theguide sequence to be tested and a control guide sequence different fromthe test guide sequence, and comparing binding or rate of cleavage at orin the vicinity of the target sequence between the test and controlguide sequence reactions. Other assays are possible, and will occur tothose skilled in the art. A guide sequence, and hence a nucleicacid-targeting guide RNA may be selected to target any target nucleicacid sequence.

In certain embodiments, the guide sequence or spacer length of the guidemolecules is from 15 to 50 nt. In certain embodiments, the spacer lengthof the guide RNA is at least 15 nucleotides. In certain embodiments, thespacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23,or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt,e.g., 24, 25, 26, or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt,from 30-35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.In certain example embodiment, the guide sequence is 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39 40, 41, 42, 43, 44, 45, 46, 47 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100 nt.

In some embodiments, the guide sequence is an RNA sequence of between 10to 50 nt in length, but more particularly of about 20-30 ntadvantageously about 20 nt, 23-25 nt or 24 nt. The guide sequence isselected so as to ensure that it hybridizes to the target sequence. Thisis described more in detail below. Selection can encompass further stepswhich increase efficacy and specificity.

In some embodiments, the guide sequence has a canonical length (e.g.,about 15-30 nt) is used to hybridize with the target RNA or DNA. In someembodiments, a guide molecule is longer than the canonical length(e.g., >30 nt) is used to hybridize with the target RNA or DNA, suchthat a region of the guide sequence hybridizes with a region of the RNAor DNA strand outside of the Cas-guide target complex. This can be ofinterest where additional modifications, such deamination of nucleotidesis of interest. In alternative embodiments, it is of interest tomaintain the limitation of the canonical guide sequence length.

In some embodiments, the sequence of the guide molecule (direct repeatand/or spacer) is selected to reduce the degree secondary structurewithin the guide molecule. In some embodiments, about or less than about75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of thenucleotides of the nucleic acid-targeting guide RNA participate inself-complementary base pairing when optimally folded. Optimal foldingmay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g., A. R. Gruber et al., 2008,Cell 106(1): 23-24; and PA Carr and GM Church, 2009, NatureBiotechnology 27(12): 1151-62).

In some embodiments, it is of interest to reduce the susceptibility ofthe guide molecule to RNA cleavage, such as to cleavage by Cas13.Accordingly, in particular embodiments, the guide molecule is adjustedto avoid cleavage by Cas13 or other RNA-cleaving enzymes.

In certain embodiments, the guide molecule comprises non-naturallyoccurring nucleic acids and/or non-naturally occurring nucleotidesand/or nucleotide analogs, and/or chemically modifications. Preferably,these non-naturally occurring nucleic acids and non-naturally occurringnucleotides are located outside the guide sequence. Non-naturallyoccurring nucleic acids can include, for example, mixtures of naturallyand non-naturally occurring nucleotides. Non-naturally occurringnucleotides and/or nucleotide analogs may be modified at the ribose,phosphate, and/or base moiety. In an embodiment of the invention, aguide nucleic acid comprises ribonucleotides and non-ribonucleotides. Inone such embodiment, a guide comprises one or more ribonucleotides andone or more deoxyribonucleotides. In an embodiment of the invention, theguide comprises one or more non-naturally occurring nucleotide ornucleotide analog such as a nucleotide with phosphorothioate linkage, alocked nucleic acid (LNA) nucleotides comprising a methylene bridgebetween the 2′ and 4′ carbons of the ribose ring, or bridged nucleicacids (BNA). Other examples of modified nucleotides include 2′-O-methylanalogs, 2′-deoxy analogs, or 2′-fluoro analogs. Further examples ofmodified bases include, but are not limited to, 2-aminopurine,5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. Examples ofguide RNA chemical modifications include, without limitation,incorporation of 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS),S-constrained ethyl(cEt), or 2′-O-methyl 3′thioPACE (MSP) at one or moreterminal nucleotides. Such chemically modified guides can compriseincreased stability and increased activity as compared to unmodifiedguides, though on-target vs. off-target specificity is not predictable.(See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290,published online 29 Jun. 2015 Ragdarm et al., 0215, PNAS, E7110-E7111;Allerson et al., J. Med Chem. 2005, 48:901-904; Bramsen et al., Front.Genet., 2012, 3:154; Deng et al., PNAS, 2015, 112:11870-11875; Sharma etal., MedChemComm., 2014, 5:1454-1471; Hendel et al., Nat. Biotechnol.(2015) 33(9): 985-989; Li et al., Nature Biomedical Engineering, 2017,1, 0066 DOI:10.1038/s41551-017-0066). In some embodiments, the 5′ and/or3′ end of a guide RNA is modified by a variety of functional moietiesincluding fluorescent dyes, polyethylene glycol, cholesterol, proteins,or detection tags. (See Kelly et al., 2016, J. Biotech. 233:74-83). Incertain embodiments, a guide comprises ribonucleotides in a region thatbinds to a target RNA and one or more deoxyribonucletides and/ornucleotide analogs in a region that binds to Cas13. In an embodiment ofthe invention, deoxyribonucleotides and/or nucleotide analogs areincorporated in engineered guide structures, such as, withoutlimitation, stem-loop regions, and the seed region. For Cas13 guide, incertain embodiments, the modification is not in the 5′-handle of thestem-loop regions. Chemical modification in the 5′-handle of thestem-loop region of a guide may abolish its function (see Li, et al.,Nature Biomedical Engineering, 2017, 1:0066). In certain embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75nucleotides of a guide is chemically modified. In some embodiments, 3-5nucleotides at either the 3′ or the 5′ end of a guide is chemicallymodified. In some embodiments, only minor modifications are introducedin the seed region, such as 2′-F modifications. In some embodiments,2′-F modification is introduced at the 3′ end of a guide. In certainembodiments, three to five nucleotides at the 5′ and/or the 3′ end ofthe guide are chemically modified with 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), S-constrained ethyl(cEt), or 2′-O-methyl 3′thioPACE (MSP). Such modification can enhance genome editing efficiency(see Hendel et al., Nat. Biotechnol. (2015) 33(9): 985-989). In certainembodiments, all of the phosphodiester bonds of a guide are substitutedwith phosphorothioates (PS) for enhancing levels of gene disruption. Incertain embodiments, more than five nucleotides at the 5′ and/or the 3′end of the guide are chemically modified with 2′-O-Me, 2′-F orS-constrained ethyl(cEt). Such chemically modified guide can mediateenhanced levels of gene disruption (see Ragdarm et al., 0215, PNAS,E7110-E7111). In an embodiment of the invention, a guide is modified tocomprise a chemical moiety at its 3′ and/or 5′ end. Such moietiesinclude, but are not limited to amine, azide, alkyne, thio,dibenzocyclooctyne (DBCO), or Rhodamine. In certain embodiment, thechemical moiety is conjugated to the guide by a linker, such as an alkylchain. In certain embodiments, the chemical moiety of the modified guidecan be used to attach the guide to another molecule, such as DNA, RNA,protein, or nanoparticles. Such chemically modified guide can be used toidentify or enrich cells generically edited by a CRISPR system (see Leeet al., eLife, 2017, 6:e25312, DOI:10.7554).

In some embodiments, the modification to the guide is a chemicalmodification, an insertion, a deletion or a split. In some embodiments,the chemical modification includes, but is not limited to, incorporationof 2′-O-methyl (M) analogs, 2′-deoxy analogs, 2-thiouridine analogs,N6-methyladenosine analogs, 2′-fluoro analogs, 2-aminopurine,5-bromo-uridine, pseudouridine (Ψ), N1-methylpseudouridine (mel1Ψ),5-methoxyuridine(5moU), inosine, 7-methylguanosine, 2′-O-methyl3′phosphorothioate (MS), S-constrained ethyl(cEt), phosphorothioate(PS), or 2′-O-methyl 3′thioPACE (MSP). In some embodiments, the guidecomprises one or more of phosphorothioate modifications. In certainembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or 25 nucleotides of the guide are chemicallymodified. In certain embodiments, one or more nucleotides in the seedregion are chemically modified. In certain embodiments, one or morenucleotides in the 3′-terminus are chemically modified. In certainembodiments, none of the nucleotides in the 5′-handle is chemicallymodified. In some embodiments, the chemical modification in the seedregion is a minor modification, such as incorporation of a 2′-fluoroanalog. In a specific embodiment, one nucleotide of the seed region isreplaced with a 2′-fluoro analog. In some embodiments, 5 to 10nucleotides in the 3′-terminus are chemically modified. Such chemicalmodifications at the 3′-terminus of the Cas13 CrRNA may improve Cas13activity. In a specific embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10nucleotides in the 3′-terminus are replaced with 2′-fluoro analogues. Ina specific embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides inthe 3′-terminus are replaced with 2′-O-methyl (M) analogs.

In some embodiments, the loop of the 5′-handle of the guide is modified.In some embodiments, the loop of the 5′-handle of the guide is modifiedto have a deletion, an insertion, a split, or chemical modifications. Incertain embodiments, the modified loop comprises 3, 4, or 5 nucleotides.In certain embodiments, the loop comprises the sequence of UCUU, UUUU,UAUU, or UGUU.

In some embodiments, the guide molecule forms a stemloop with a separatenon-covalently linked sequence, which can be DNA or RNA. In particularembodiments, the sequences forming the guide are first synthesized usingthe standard phosphoramidite synthetic protocol (Herdewijn, P., ed.,Methods in Molecular Biology Col 288, Oligonucleotide Synthesis: Methodsand Applications, Humana Press, New Jersey (2012)). In some embodiments,these sequences can be functionalized to contain an appropriatefunctional group for ligation using the standard protocol known in theart (Hermanson, G. T., Bioconjugate Techniques, Academic Press (2013)).Examples of functional groups include, but are not limited to, hydroxyl,amine, carboxylic acid, carboxylic acid halide, carboxylic acid activeester, aldehyde, carbonyl, chlorocarbonyl, imidazolylcarbonyl,hydrozide, semicarbazide, thio semicarbazide, thiol, maleimide,haloalkyl, sufonyl, ally, propargyl, diene, alkyne, and azide. Once thissequence is functionalized, a covalent chemical bond or linkage can beformed between this sequence and the direct repeat sequence. Examples ofchemical bonds include, but are not limited to, those based oncarbamates, ethers, esters, amides, imines, amidines, aminotrizines,hydrozone, disulfides, thioethers, thioesters, phosphorothioates,phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides,ureas, thioureas, hydrazide, oxime, triazole, photolabile linkages, C—Cbond forming groups such as Diels-Alder cyclo-addition pairs orring-closing metathesis pairs, and Michael reaction pairs.

In some embodiments, these stem-loop forming sequences can be chemicallysynthesized. In some embodiments, the chemical synthesis uses automated,solid-phase oligonucleotide synthesis machines with 2′-acetoxyethylorthoester (2′-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18) or2′-thionocarbamate (2′-TC) chemistry (Dellinger et al., J. Am. Chem.Soc. (2011) 133: 11540-11546; Hendel et al., Nat. Biotechnol. (2015)33:985-989).

In certain embodiments, the guide molecule comprises (1) a guidesequence capable of hybridizing to a target locus and (2) a tracr mateor direct repeat sequence whereby the direct repeat sequence is locatedupstream (i.e., 5′) from the guide sequence. In a particular embodimentthe seed sequence (i.e. the sequence essential critical for recognitionand/or hybridization to the sequence at the target locus) of the guidesequence is approximately within the first 10 nucleotides of the guidesequence.

In a particular embodiment the guide molecule comprises a guide sequencelinked to a direct repeat sequence, wherein the direct repeat sequencecomprises one or more stem loops or optimized secondary structures. Inparticular embodiments, the direct repeat has a minimum length of 16 ntsand a single stem loop. In further embodiments the direct repeat has alength longer than 16 nts, preferably more than 17 nts, and has morethan one stem loops or optimized secondary structures. In particularembodiments the guide molecule comprises or consists of the guidesequence linked to all or part of the natural direct repeat sequence. Atypical Type V or Type VI CRISPR-cas guide molecule comprises (in 3′ to5′ direction or in 5′ to 3′ direction): a guide sequence a firstcomplimentary stretch (the “repeat”), a loop (which is typically 4 or 5nucleotides long), a second complimentary stretch (the “anti-repeat”being complimentary to the repeat), and a poly A (often poly U in RNA)tail (terminator). In certain embodiments, the direct repeat sequenceretains its natural architecture and forms a single stem loop. Inparticular embodiments, certain aspects of the guide architecture can bemodified, for example by addition, subtraction, or substitution offeatures, whereas certain other aspects of guide architecture aremaintained. Preferred locations for engineered guide moleculemodifications, including but not limited to insertions, deletions, andsubstitutions include guide termini and regions of the guide moleculethat are exposed when complexed with the CRISPR-Cas protein and/ortarget, for example the stemloop of the direct repeat sequence.

In particular embodiments, the stem comprises at least about 4 bpcomprising complementary X and Y sequences, although stems of more,e.g., 5, 6, 7, 8, 9, 10, 11 or 12 or fewer, e.g., 3, 2, base pairs arealso contemplated. Thus, for example X2-10 and Y2-10 (wherein X and Yrepresent any complementary set of nucleotides) may be contemplated. Inone aspect, the stem made of the X and Y nucleotides, together with theloop will form a complete hairpin in the overall secondary structure;and, this may be advantageous and the amount of base pairs can be anyamount that forms a complete hairpin. In one aspect, any complementaryX:Y basepairing sequence (e.g., as to length) is tolerated, so long asthe secondary structure of the entire guide molecule is preserved. Inone aspect, the loop that connects the stem made of X:Y basepairs can beany sequence of the same length (e.g., 4 or 5 nucleotides) or longerthat does not interrupt the overall secondary structure of the guidemolecule. In one aspect, the stemloop can further comprise, e.g. an MS2aptamer. In one aspect, the stem comprises about 5-7 bp comprisingcomplementary X and Y sequences, although stems of more or fewerbasepairs are also contemplated. In one aspect, non-Watson Crickbasepairing is contemplated, where such pairing otherwise generallypreserves the architecture of the stemloop at that position.

In particular embodiments the natural hairpin or stemloop structure ofthe guide molecule is extended or replaced by an extended stemloop. Ithas been demonstrated that extension of the stem can enhance theassembly of the guide molecule with the CRISPR-Cas proten (Chen et al.Cell. (2013); 155(7): 1479-1491). In particular embodiments the stem ofthe stemloop is extended by at least 1, 2, 3, 4, 5 or more complementarybasepairs (i.e. corresponding to the addition of 2, 4, 6, 8, 10 or morenucleotides in the guide molecule). In particular embodiments these arelocated at the end of the stem, adjacent to the loop of the stemloop.

In particular embodiments, the susceptibility of the guide molecule toRNAses or to decreased expression can be reduced by slight modificationsof the sequence of the guide molecule which do not affect its function.For instance, in particular embodiments, premature termination oftranscription, such as premature transcription of U6 Pol-III, can beremoved by modifying a putative Pol-III terminator (4 consecutive U's)in the guide molecules sequence. Where such sequence modification isrequired in the stemloop of the guide molecule, it is preferably ensuredby a basepair flip.

In a particular embodiment the direct repeat may be modified to compriseone or more protein-binding RNA aptamers. In a particular embodiment,one or more aptamers may be included such as part of optimized secondarystructure. Such aptamers may be capable of binding a bacteriophage coatprotein as detailed further herein.

In some embodiments, the guide molecule forms a duplex with a target RNAcomprising at least one target cytosine residue to be edited. Uponhybridization of the guide RNA molecule to the target RNA, the cytidinedeaminase binds to the single strand RNA in the duplex made accessibleby the mismatch in the guide sequence and catalyzes deamination of oneor more target cytosine residues comprised within the stretch ofmismatching nucleotides.

A guide sequence, and hence a nucleic acid-targeting guide RNA may beselected to target any target nucleic acid sequence. The target sequencemay be mRNA.

In certain embodiments, the target sequence should be associated with aPAM (protospacer adjacent motif) or PFS (protospacer flanking sequenceor site); that is, a short sequence recognized by the CRISPR complex.Depending on the nature of the CRISPR-Cas protein, the target sequenceshould be selected such that its complementary sequence in the DNAduplex (also referred to herein as the non-target sequence) is upstreamor downstream of the PAM. In the embodiments of the present inventionwhere the CRISPR-Cas protein is a Cas13 protein, the complementarysequence of the target sequence is downstream or 3′ of the PAM orupstream or 5′ of the PAM. The precise sequence and length requirementsfor the PAM differ depending on the Cas13 protein used, but PAMs aretypically 2-5 base pair sequences adjacent the protospacer (that is, thetarget sequence). Examples of the natural PAM sequences for differentCas13 orthologues are provided herein below and the skilled person willbe able to identify further PAM sequences for use with a given Cas13protein.

Further, engineering of the PAM Interacting (PI) domain may allowprograming of PAM specificity, improve target site recognition fidelity,and increase the versatility of the CRISPR-Cas protein, for example asdescribed for Cas9 in Kleinstiver B P et al. Engineered CRISPR-Cas9nucleases with altered PAM specificities. Nature. 2015 Jul. 23;523(7561):481-5. doi: 10.1038/nature14592. As further detailed herein,the skilled person will understand that Cas13 proteins may be modifiedanalogously.

In particular embodiment, the guide is an escorted guide. By “escorted”is meant that the CRISPR-Cas system or complex or guide is delivered toa selected time or place within a cell, so that activity of theCRISPR-Cas system or complex or guide is spatially or temporallycontrolled. For example, the activity and destination of the 3CRISPR-Cas system or complex or guide may be controlled by an escort RNAaptamer sequence that has binding affinity for an aptamer ligand, suchas a cell surface protein or other localized cellular component.Alternatively, the escort aptamer may for example be responsive to anaptamer effector on or in the cell, such as a transient effector, suchas an external energy source that is applied to the cell at a particulartime.

The escorted CRISPR-Cas systems or complexes have a guide molecule witha functional structure designed to improve guide molecule structure,architecture, stability, genetic expression, or any combination thereofSuch a structure can include an aptamer.

Aptamers are biomolecules that can be designed or selected to bindtightly to other ligands, for example using a technique calledsystematic evolution of ligands by exponential enrichment (SELEX; TuerkC, Gold L: “Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4 DNA polymerase.” Science 1990,249:505-510). Nucleic acid aptamers can for example be selected frompools of random-sequence oligonucleotides, with high binding affinitiesand specificities for a wide range of biomedically relevant targets,suggesting a wide range of therapeutic utilities for aptamers (Keefe,Anthony D., Supriya Pai, and Andrew Ellington. “Aptamers astherapeutics.” Nature Reviews Drug Discovery 9.7 (2010): 537-550). Thesecharacteristics also suggest a wide range of uses for aptamers as drugdelivery vehicles (Levy-Nissenbaum, Etgar, et al. “Nanotechnology andaptamers: applications in drug delivery.” Trends in biotechnology 26.8(2008): 442-449; and, Hicke B J, Stephens A W. “Escort aptamers: adelivery service for diagnosis and therapy.” J Clin Invest 2000,106:923-928.). Aptamers may also be constructed that function asmolecular switches, responding to a que by changing properties, such asRNA aptamers that bind fluorophores to mimic the activity of greenflourescent protein (Paige, Jeremy S., Karen Y. Wu, and Samie R.Jaffrey. “RNA mimics of green fluorescent protein.” Science 333.6042(2011): 642-646). It has also been suggested that aptamers may be usedas components of targeted siRNA therapeutic delivery systems, forexample targeting cell surface proteins (Zhou, Jiehua, and John J.Rossi. “Aptamer-targeted cell-specific RNA interference.” Silence 1.1(2010): 4).

Accordingly, in particular embodiments, the guide molecule is modified,e.g., by one or more aptamer(s) designed to improve guide moleculedelivery, including delivery across the cellular membrane, tointracellular compartments, or into the nucleus. Such a structure caninclude, either in addition to the one or more aptamer(s) or withoutsuch one or more aptamer(s), moiety(ies) so as to render the guidemolecule deliverable, inducible or responsive to a selected effector.The invention accordingly comprehends a guide molecule that responds tonormal or pathological physiological conditions, including withoutlimitation pH, hypoxia, O₂ concentration, temperature, proteinconcentration, enzymatic concentration, lipid structure, light exposure,mechanical disruption (e.g. ultrasound waves), magnetic fields, electricfields, or electromagnetic radiation.

Light responsiveness of an inducible system may be achieved via theactivation and binding of cryptochrome-2 and CIB1. Blue lightstimulation induces an activating conformational change incryptochrome-2, resulting in recruitment of its binding partner CIB1.This binding is fast and reversible, achieving saturation in <15 secfollowing pulsed stimulation and returning to baseline <15 min after theend of stimulation. These rapid binding kinetics result in a systemtemporally bound only by the speed of transcription/translation andtranscript/protein degradation, rather than uptake and clearance ofinducing agents. Crytochrome-2 activation is also highly sensitive,allowing for the use of low light intensity stimulation and mitigatingthe risks of phototoxicity. Further, in a context such as the intactmammalian brain, variable light intensity may be used to control thesize of a stimulated region, allowing for greater precision than vectordelivery alone may offer.

The invention contemplates energy sources such as electromagneticradiation, sound energy or thermal energy to induce the guide.Advantageously, the electromagnetic radiation is a component of visiblelight. In a preferred embodiment, the light is a blue light with awavelength of about 450 to about 495 nm. In an especially preferredembodiment, the wavelength is about 488 nm. In another preferredembodiment, the light stimulation is via pulses. The light power mayrange from about 0-9 mW/cm². In a preferred embodiment, a stimulationparadigm of as low as 0.25 sec every 15 sec should result in maximalactivation.

The chemical or energy sensitive guide may undergo a conformationalchange upon induction by the binding of a chemical source or by theenergy allowing it act as a guide and have the Cas13 CRISPR-Cas systemor complex function. The invention can involve applying the chemicalsource or energy so as to have the guide function and the Cas13CRISPR-Cas system or complex function; and optionally furtherdetermining that the expression of the genomic locus is altered.

There are several different designs of this chemical induciblesystem: 1. ABI-PYL based system inducible by Abscisic Acid (ABA) (see,e.g.,http://stke.sciencemag.org/cgi/content/abstract/sigtrans;4/164/rs2), 2.FKBP-FRB based system inducible by rapamycin (or related chemicals basedon rapamycin) (see, e.g.,http://www.nature.com/nmeth/journal/v2/n6/full/nmeth763.html), 3.GID1-GAI based system inducible by Gibberellin (GA) (see, e.g.,http://www.nature.com/nchembio/journal/v8/n5/full/nchembio.922.html).

A chemical inducible system can be an estrogen receptor (ER) basedsystem inducible by 4-hydroxytamoxifen (4OHT) (see, e.g.,http://www.pnas.org/content/I04/3/1027.abstract). A mutatedligand-binding domain of the estrogen receptor called ERT2 translocatesinto the nucleus of cells upon binding of 4-hydroxytamoxifen. In furtherembodiments of the invention any naturally occurring or engineeredderivative of any nuclear receptor, thyroid hormone receptor, retinoicacid receptor, estrogren receptor, estrogen-related receptor,glucocorticoid receptor, progesterone receptor, androgen receptor may beused in inducible systems analogous to the ER based inducible system.

Another inducible system is based on the design using Transient receptorpotential (TRP) ion channel based system inducible by energy, heat orradio-wave (see, e.g., http://www.sciencemag.org/content/336/6081/604).These TRP family proteins respond to different stimuli, including lightand heat. When this protein is activated by light or heat, the ionchannel will open and allow the entering of ions such as calcium intothe plasma membrane. This influx of ions will bind to intracellular ioninteracting partners linked to a polypeptide including the guide and theother components of the Cas13 CRISPR-Cas complex or system, and thebinding will induce the change of sub-cellular localization of thepolypeptide, leading to the entire polypeptide entering the nucleus ofcells. Once inside the nucleus, the guide protein and the othercomponents of the Cas13 CRISPR-Cas complex will be active and modulatingtarget gene expression in cells.

While light activation may be an advantageous embodiment, sometimes itmay be disadvantageous especially for in vivo applications in which thelight may not penetrate the skin or other organs. In this instance,other methods of energy activation are contemplated, in particular,electric field energy and/or ultrasound which have a similar effect.

Electric field energy is preferably administered substantially asdescribed in the art, using one or more electric pulses of from about 1Volt/cm to about 10 kVolts/cm under in vivo conditions. Instead of or inaddition to the pulses, the electric field may be delivered in acontinuous manner. The electric pulse may be applied for between 1 psand 500 milliseconds, preferably between 1 ps and 100 milliseconds. Theelectric field may be applied continuously or in a pulsed manner for 5about minutes.

As used herein, ‘electric field energy’ is the electrical energy towhich a cell is exposed. Preferably the electric field has a strength offrom about 1 Volt/cm to about 10 kVolts/cm or more under in vivoconditions (see WO97/49450).

As used herein, the term “electric field” includes one or more pulses atvariable capacitance and voltage and including exponential and/or squarewave and/or modulated wave and/or modulated square wave forms.References to electric fields and electricity should be taken to includereference the presence of an electric potential difference in theenvironment of a cell. Such an environment may be set up by way ofstatic electricity, alternating current (AC), direct current (DC), etc,as known in the art. The electric field may be uniform, non-uniform orotherwise, and may vary in strength and/or direction in a time dependentmanner.

Single or multiple applications of electric field, as well as single ormultiple applications of ultrasound are also possible, in any order andin any combination. The ultrasound and/or the electric field may bedelivered as single or multiple continuous applications, or as pulses(pulsatile delivery).

Electroporation has been used in both in vitro and in vivo procedures tointroduce foreign material into living cells. With in vitroapplications, a sample of live cells is first mixed with the agent ofinterest and placed between electrodes such as parallel plates. Then,the electrodes apply an electrical field to the cell/implant mixture.Examples of systems that perform in vitro electroporation include theElectro Cell Manipulator ECM600 product, and the Electro Square PoratorT820, both made by the BTX Division of Genetronics, Inc (see U.S. Pat.No. 5,869,326).

The known electroporation techniques (both in vitro and in vivo)function by applying a brief high voltage pulse to electrodes positionedaround the treatment region. The electric field generated between theelectrodes causes the cell membranes to temporarily become porous,whereupon molecules of the agent of interest enter the cells. In knownelectroporation applications, this electric field comprises a singlesquare wave pulse on the order of 1000 V/cm, of about 100 .mu.sduration. Such a pulse may be generated, for example, in knownapplications of the Electro Square Porator T820.

Preferably, the electric field has a strength of from about 1 V/cm toabout 10 kV/cm under in vitro conditions. Thus, the electric field mayhave a strength of 1 V/cm, 2 V/cm, 3 V/cm, 4 V/cm, 5 V/cm, 6 V/cm, 7V/cm, 8 V/cm, 9 V/cm, 10 V/cm, 20 V/cm, 50 V/cm, 100 V/cm, 200 V/cm, 300V/cm, 400 V/cm, 500 V/cm, 600 V/cm, 700 V/cm, 800 V/cm, 900 V/cm, 1kV/cm, 2 kV/cm, 5 kV/cm, 10 kV/cm, 20 kV/cm, 50 kV/cm or more. Morepreferably from about 0.5 kV/cm to about 4.0 kV/cm under in vitroconditions. Preferably the electric field has a strength of from about 1V/cm to about 10 kV/cm under in vivo conditions. However, the electricfield strengths may be lowered where the number of pulses delivered tothe target site are increased. Thus, pulsatile delivery of electricfields at lower field strengths is envisaged.

Preferably the application of the electric field is in the form ofmultiple pulses such as double pulses of the same strength andcapacitance or sequential pulses of varying strength and/or capacitance.As used herein, the term “pulse” includes one or more electric pulses atvariable capacitance and voltage and including exponential and/or squarewave and/or modulated wave/square wave forms.

Preferably the electric pulse is delivered as a waveform selected froman exponential wave form, a square wave form, a modulated wave form anda modulated square wave form.

A preferred embodiment employs direct current at low voltage. Thus,Applicants disclose the use of an electric field which is applied to thecell, tissue or tissue mass at a field strength of between 1V/cm and20V/cm, for a period of 100 milliseconds or more, preferably 15 minutesor more.

Ultrasound is advantageously administered at a power level of from about0.05 W/cm2 to about 100 W/cm2. Diagnostic or therapeutic ultrasound maybe used, or combinations thereof.

As used herein, the term “ultrasound” refers to a form of energy whichconsists of mechanical vibrations the frequencies of which are so highthey are above the range of human hearing. Lower frequency limit of theultrasonic spectrum may generally be taken as about 20 kHz. Mostdiagnostic applications of ultrasound employ frequencies in the range 1and 15 MHz′ (From Ultrasonics in Clinical Diagnosis, P. N. T. Wells,ed., 2nd. Edition, Publ. Churchill Livingstone [Edinburgh, London & NY,1977]).

Ultrasound has been used in both diagnostic and therapeuticapplications. When used as a diagnostic tool (“diagnostic ultrasound”),ultrasound is typically used in an energy density range of up to about100 mW/cm2 (FDA recommendation), although energy densities of up to 750mW/cm2 have been used. In physiotherapy, ultrasound is typically used asan energy source in a range up to about 3 to 4 W/cm2 (WHOrecommendation). In other therapeutic applications, higher intensitiesof ultrasound may be employed, for example, HIFU at 100 W/cm up to 1kW/cm2 (or even higher) for short periods of time. The term “ultrasound”as used in this specification is intended to encompass diagnostic,therapeutic and focused ultrasound.

Focused ultrasound (FUS) allows thermal energy to be delivered withoutan invasive probe (see Morocz et al 1998 Journal of Magnetic ResonanceImaging Vol. 8, No. 1, pp. 136-142. Another form of focused ultrasoundis high intensity focused ultrasound (HIFU) which is reviewed byMoussatov et al in Ultrasonics (1998) Vol. 36, No. 8, pp. 893-900 andTranHuuHue et al in Acustica (1997) Vol. 83, No. 6, pp. 1103-1106.

Preferably, a combination of diagnostic ultrasound and a therapeuticultrasound is employed. This combination is not intended to be limiting,however, and the skilled reader will appreciate that any variety ofcombinations of ultrasound may be used. Additionally, the energydensity, frequency of ultrasound, and period of exposure may be varied.

Preferably the exposure to an ultrasound energy source is at a powerdensity of from about 0.05 to about 100 Wcm-2. Even more preferably, theexposure to an ultrasound energy source is at a power density of fromabout 1 to about 15 Wcm-2.

Preferably the exposure to an ultrasound energy source is at a frequencyof from about 0.015 to about 10.0 MHz. More preferably the exposure toan ultrasound energy source is at a frequency of from about 0.02 toabout 5.0 MHz or about 6.0 MHz. Most preferably, the ultrasound isapplied at a frequency of 3 MHz.

Preferably the exposure is for periods of from about 10 milliseconds toabout 60 minutes. Preferably the exposure is for periods of from about 1second to about 5 minutes. More preferably, the ultrasound is appliedfor about 2 minutes. Depending on the particular target cell to bedisrupted, however, the exposure may be for a longer duration, forexample, for 15 minutes.

Advantageously, the target tissue is exposed to an ultrasound energysource at an acoustic power density of from about 0.05 Wcm-2 to about 10Wcm-2 with a frequency ranging from about 0.015 to about 10 MHz (see WO98/52609). However, alternatives are also possible, for example,exposure to an ultrasound energy source at an acoustic power density ofabove 100 Wcm-2, but for reduced periods of time, for example, 1000Wcm-2 for periods in the millisecond range or less.

Preferably the application of the ultrasound is in the form of multiplepulses; thus, both continuous wave and pulsed wave (pulsatile deliveryof ultrasound) may be employed in any combination. For example,continuous wave ultrasound may be applied, followed by pulsed waveultrasound, or vice versa. This may be repeated any number of times, inany order and combination. The pulsed wave ultrasound may be appliedagainst a background of continuous wave ultrasound, and any number ofpulses may be used in any number of groups.

Preferably, the ultrasound may comprise pulsed wave ultrasound. In ahighly preferred embodiment, the ultrasound is applied at a powerdensity of 0.7 Wcm-2 or 1.25 Wcm-2 as a continuous wave. Higher powerdensities may be employed if pulsed wave ultrasound is used.

Use of ultrasound is advantageous as, like light, it may be focusedaccurately on a target. Moreover, ultrasound is advantageous as it maybe focused more deeply into tissues unlike light. It is therefore bettersuited to whole-tissue penetration (such as but not limited to a lobe ofthe liver) or whole organ (such as but not limited to the entire liveror an entire muscle, such as the heart) therapy. Another importantadvantage is that ultrasound is a non-invasive stimulus which is used ina wide variety of diagnostic and therapeutic applications. By way ofexample, ultrasound is well known in medical imaging techniques and,additionally, in orthopedic therapy. Furthermore, instruments suitablefor the application of ultrasound to a subject vertebrate are widelyavailable and their use is well known in the art.

In particular embodiments, the guide molecule is modified by a secondarystructure to increase the specificity of the CRISPR-Cas system and thesecondary structure can protect against exonuclease activity and allowfor 5′ additions to the guide sequence also referred to herein as aprotected guide molecule.

In one aspect, the invention provides for hybridizing a “protector RNA”to a sequence of the guide molecule, wherein the “protector RNA” is anRNA strand complementary to the 3′ end of the guide molecule to therebygenerate a partially double-stranded guide RNA. In an embodiment of theinvention, protecting mismatched bases (i.e. the bases of the guidemolecule which do not form part of the guide sequence) with a perfectlycomplementary protector sequence decreases the likelihood of target RNAbinding to the mismatched basepairs at the 3′ end. In particularembodiments of the invention, additional sequences comprising anextented length may also be present within the guide molecule such thatthe guide comprises a protector sequence within the guide molecule. This“protector sequence” ensures that the guide molecule comprises a“protected sequence” in addition to an “exposed sequence” (comprisingthe part of the guide sequence hybridizing to the target sequence). Inparticular embodiments, the guide molecule is modified by the presenceof the protector guide to comprise a secondary structure such as ahairpin. Advantageously there are three or four to thirty or more, e.g.,about 10 or more, contiguous base pairs having complementarity to theprotected sequence, the guide sequence or both. It is advantageous thatthe protected portion does not impede thermodynamics of the CRISPR-Cassystem interacting with its target. By providing such an extensionincluding a partially double stranded guide molecule, the guide moleculeis considered protected and results in improved specific binding of theCRISPR-Cas complex, while maintaining specific activity.

In particular embodiments, use is made of a truncated guide (tru-guide),i.e. a guide molecule which comprises a guide sequence which istruncated in length with respect to the canonical guide sequence length.As described by Nowak et al. (Nucleic Acids Res (2016) 44 (20):9555-9564), such guides may allow catalytically active CRISPR-Cas enzymeto bind its target without cleaving the target RNA. In particularembodiments, a truncated guide is used which allows the binding of thetarget but retains only nickase activity of the CRISPR-Cas enzyme.

The present invention may be further illustrated and extended based onaspects of CRISPR-Cas development and use as set forth in the followingarticles and particularly as relates to delivery of a CRISPR proteincomplex and uses of an RNA guided endonuclease in cells and organisms:

-   Multiplex genome engineering using CRISPR-Cas systems. Cong, L.,    Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D.,    Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science February    15; 339(6121):819-23 (2013);-   RNA-guided editing of bacterial genomes using CRISPR-Cas systems.    Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A. Nat Biotechnol    March;31(3):233-9 (2013);-   One-Step Generation of Mice Carrying Mutations in Multiple Genes by    CRISPR-Cas-Mediated Genome Engineering. Wang H., Yang H., Shivalila    C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R. Cell May 9;    153(4):910-8 (2013);-   Optical control of mammalian endogenous transcription and epigenetic    states. Konermann S, Brigham M D, Trevino A E, Hsu P D, Heidenreich    M, Cong L, Platt R J, Scott D A, Church G M, Zhang F. Nature. August    22; 500(7463):472-6. doi: 10.1038/Nature12466. Epub 2013 Aug. 23    (2013);-   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing    Specificity. Ran, F A., Hsu, P D., Lin, C Y., Gootenberg, J S.,    Konermann, S., Trevino, A E., Scott, D A., Inoue, A., Matoba, S.,    Zhang, Y., & Zhang, F. Cell August 28. pii: S0092-8674(13)01015-5    (2013-A);-   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,    Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V.,    Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L    A., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013);-   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu, P    D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature    Protocols November;8(11):2281-308 (2013-B);-   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem,    O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson,    T., Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F.    Science December 12. (2013);-   Crystal structure of cas9 in complex with guide RNA and target DNA.    Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S., Shehata, S I.,    Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell February 27,    156(5):935-49 (2014);-   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian    cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon D    B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch R.,    Zhang F., Sharp P A. Nat Biotechnol. April 20. doi: 10.1038/nbt.2889    (2014);-   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.    Platt R J, Chen S, Zhou Y, Yim M J, Swiech L, Kempton H R, Dahlman J    E, Parnas O, Eisenhaure T M, Jovanovic M, Graham D B, Jhunjhunwala    S, Heidenreich M, Xavier R J, Langer R, Anderson D G, Hacohen N,    Regev A, Feng G, Sharp P A, Zhang F. Cell 159(2): 440-455 DOI:    10.1016/j.cell.2014.09.014(2014);-   Development and Applications of CRISPR-Cas9 for Genome Engineering,    Hsu P D, Lander E S, Zhang F., Cell. June 5; 157(6):1262-78 (2014).-   Genetic screens in human cells using the CRISPR-Cas9 system, Wang T,    Wei J J, Sabatini D M, Lander E S., Science. January 3; 343(6166):    80-84. doi:10.1126/science.1246981 (2014);-   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated    gene inactivation, Doench J G, Hartenian E, Graham D B, Tothova Z,    Hegde M, Smith I, Sullender M, Ebert B L, Xavier R J, Root D E.,    (published online 3 Sep. 2014) Nat Biotechnol. December;    32(12):1262-7 (2014);-   In vivo interrogation of gene function in the mammalian brain using    CRISPR-Cas9, Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y,    Trombetta J, Sur M, Zhang F., (published online 19 Oct. 2014) Nat    Biotechnol. January;33(1):102-6 (2015);-   Genome-scale transcriptional activation by an engineered CRISPR-Cas9    complex, Konermann S, Brigham M D, Trevino A E, Joung J, Abudayyeh O    O, Barcena C, Hsu P D, Habib N, Gootenberg J S, Nishimasu H, Nureki    O, Zhang F., Nature. January 29; 517(7536):583-8 (2015).-   A split-Cas9 architecture for inducible genome editing and    transcription modulation, Zetsche B, Volz S E, Zhang F., (published    online 2 Feb. 2015) Nat Biotechnol. February;33(2):139-42 (2015);-   Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and    Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi X,    Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F, Sharp P A.    Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen in mouse), and-   In vivo genome editing using Staphylococcus aureus Cas9, Ran F A,    Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche B,    Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang F.,    (published online 1 Apr. 2015), Nature. April 9;    520(7546):186-91(2015).-   Shalem et al., “High-throughput functional genomics using    CRISPR-Cas9,” Nature Reviews Genetics 16, 299-311 (May 2015).-   Xu et al., “Sequence determinants of improved CRISPR sgRNA design,”    Genome Research 25, 1147-1157 (August 2015).-   Parnas et al., “A Genome-wide CRISPR Screen in Primary Immune Cells    to Dissect Regulatory Networks,” Cell 162, 675-686 (Jul. 30, 2015).-   Ramanan et al., CRISPR-Cas9 cleavage of viral DNA efficiently    suppresses hepatitis B virus,” Scientific Reports 5:10833. doi:    10.1038/srep10833 (Jun. 2, 2015)-   Nishimasu et al., Crystal Structure of Staphylococcus aureus Cas9,”    Cell 162, 1113-1126 (Aug. 27, 2015)-   BCL11A enhancer dissection by Cas9-mediated in situ saturating    mutagenesis, Canver et al., Nature 527(7577):192-7 (Nov. 12, 2015)    doi: 10.1038/nature15521. Epub 2015 Sep. 16.-   Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas    System, Zetsche et al., Cell 163, 759-71 (Sep. 25, 2015).-   Discovery and Functional Characterization of Diverse Class 2    CRISPR-Cas Systems, Shmakov et al., Molecular Cell, 60(3), 385-397    doi: 10.1016/j.molcel.2015.10.008 Epub Oct. 22, 2015.-   Rationally engineered Cas9 nucleases with improved specificity,    Slaymaker et al., Science 2016 Jan. 1 351(6268): 84-88 doi:    10.1126/science.aad5227. Epub 2015 Dec. 1.-   Gao et al, “Engineered Cpf1 Enzymes with Altered PAM Specificities,”    bioRxiv 091611; doi: http://dx.doi.org/10.1101/091611 (Dec. 4,    2016).    each of which is incorporated herein by reference, may be considered    in the practice of the instant invention, and discussed briefly    below:    -   Cong et al. engineered type II CRISPR-Cas systems for use in        eukaryotic cells based on both Streptococcus thermophilus Cas9        and also Streptococcus pyogenes Cas9 and demonstrated that Cas9        nucleases can be directed by short RNAs to induce precise        cleavage of DNA in human and mouse cells. Their study further        showed that Cas9 as converted into a nicking enzyme can be used        to facilitate homology-directed repair in eukaryotic cells with        minimal mutagenic activity. Additionally, their study        demonstrated that multiple guide sequences can be encoded into a        single CRISPR array to enable simultaneous editing of several at        endogenous genomic loci sites within the mammalian genome,        demonstrating easy programmability and wide applicability of the        RNA-guided nuclease technology. This ability to use RNA to        program sequence specific DNA cleavage in cells defined a new        class of genome engineering tools. These studies further showed        that other CRISPR loci are likely to be transplantable into        mammalian cells and can also mediate mammalian genome cleavage.        Importantly, it can be envisaged that several aspects of the        CRISPR-Cas system can be further improved to increase its        efficiency and versatility.    -   Jiang et al. used the clustered, regularly interspaced, short        palindromic repeats (CRISPR)-associated Cas9 endonuclease        complexed with dual-RNAs to introduce precise mutations in the        genomes of Streptococcus pneumoniae and Escherichia coli. The        approach relied on dual-RNA:Cas9-directed cleavage at the        targeted genomic site to kill unmutated cells and circumvents        the need for selectable markers or counter-selection systems.        The study reported reprogramming dual-RNA:Cas9 specificity by        changing the sequence of short CRISPR RNA (crRNA) to make        single- and multinucleotide changes carried on editing        templates. The study showed that simultaneous use of two crRNAs        enabled multiplex mutagenesis. Furthermore, when the approach        was used in combination with recombineering, in S. pneumoniae,        nearly 100% of cells that were recovered using the described        approach contained the desired mutation, and in E. coli, 65%        that were recovered contained the mutation.    -   Wang et al. (2013) used the CRISPR-Cas system for the one-step        generation of mice carrying mutations in multiple genes which        were traditionally generated in multiple steps by sequential        recombination in embryonic stem cells and/or time-consuming        intercrossing of mice with a single mutation. The CRISPR-Cas        system will greatly accelerate the in vivo study of functionally        redundant genes and of epistatic gene interactions.    -   Konermann et al. (2013) addressed the need in the art for        versatile and robust technologies that enable optical and        chemical modulation of DNA-binding domains based CRISPR Cas9        enzyme and also Transcriptional Activator Like Effectors    -   Ran et al. (2013-A) described an approach that combined a Cas9        nickase mutant with paired guide RNAs to introduce targeted        double-strand breaks. This addresses the issue of the Cas9        nuclease from the microbial CRISPR-Cas system being targeted to        specific genomic loci by a guide sequence, which can tolerate        certain mismatches to the DNA target and thereby promote        undesired off-target mutagenesis. Because individual nicks in        the genome are repaired with high fidelity, simultaneous nicking        via appropriately offset guide RNAs is required for        double-stranded breaks and extends the number of specifically        recognized bases for target cleavage. The authors demonstrated        that using paired nicking can reduce off-target activity by 50-        to 1,500-fold in cell lines and to facilitate gene knockout in        mouse zygotes without sacrificing on-target cleavage efficiency.        This versatile strategy enables a wide variety of genome editing        applications that require high specificity.    -   Hsu et al. (2013) characterized SpCas9 targeting specificity in        human cells to inform the selection of target sites and avoid        off-target effects. The study evaluated >700 guide RNA variants        and SpCas9-induced indel mutation levels at >100 predicted        genomic off-target loci in 293T and 293FT cells. The authors        that SpCas9 tolerates mismatches between guide RNA and target        DNA at different positions in a sequence-dependent manner,        sensitive to the number, position and distribution of        mismatches. The authors further showed that SpCas9-mediated        cleavage is unaffected by DNA methylation and that the dosage of        SpCas9 and guide RNA can be titrated to minimize off-target        modification. Additionally, to facilitate mammalian genome        engineering applications, the authors reported providing a        web-based software tool to guide the selection and validation of        target sequences as well as off-target analyses.    -   Ran et al. (2013-B) described a set of tools for Cas9-mediated        genome editing via non-homologous end joining (NHEJ) or        homology-directed repair (HDR) in mammalian cells, as well as        generation of modified cell lines for downstream functional        studies. To minimize off-target cleavage, the authors further        described a double-nicking strategy using the Cas9 nickase        mutant with paired guide RNAs. The protocol provided by the        authors experimentally derived guidelines for the selection of        target sites, evaluation of cleavage efficiency and analysis of        off-target activity. The studies showed that beginning with        target design, gene modifications can be achieved within as        little as 1-2 weeks, and modified clonal cell lines can be        derived within 2-3 weeks.    -   Shalem et al. described a new way to interrogate gene function        on a genome-wide scale. Their studies showed that delivery of a        genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted        18,080 genes with 64,751 unique guide sequences enabled both        negative and positive selection screening in human cells. First,        the authors showed use of the GeCKO library to identify genes        essential for cell viability in cancer and pluripotent stem        cells. Next, in a melanoma model, the authors screened for genes        whose loss is involved in resistance to vemurafenib, a        therapeutic that inhibits mutant protein kinase BRAF. Their        studies showed that the highest-ranking candidates included        previously validated genes NF1 and MED12 as well as novel hits        NF2, CUL3, TADA2B, and TADA1. The authors observed a high level        of consistency between independent guide RNAs targeting the same        gene and a high rate of hit confirmation, and thus demonstrated        the promise of genome-scale screening with Cas9.    -   Nishimasu et al. reported the crystal structure of        Streptococcuspyogenes Cas9 in complex with sgRNA and its target        DNA at 2.5 A° resolution. The structure revealed a bilobed        architecture composed of target recognition and nuclease lobes,        accommodating the sgRNA:DNA heteroduplex in a positively charged        groove at their interface. Whereas the recognition lobe is        essential for binding sgRNA and DNA, the nuclease lobe contains        the HNH and RuvC nuclease domains, which are properly positioned        for cleavage of the complementary and non-complementary strands        of the target DNA, respectively. The nuclease lobe also contains        a carboxyl-terminal domain responsible for the interaction with        the protospacer adjacent motif (PAM). This high-resolution        structure and accompanying functional analyses have revealed the        molecular mechanism of RNA-guided DNA targeting by Cas9, thus        paving the way for the rational design of new, versatile        genome-editing technologies.    -   Wu et al. mapped genome-wide binding sites of a catalytically        inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with        single guide RNAs (sgRNAs) in mouse embryonic stem cells        (mESCs). The authors showed that each of the four sgRNAs tested        targets dCas9 to between tens and thousands of genomic sites,        frequently characterized by a 5-nucleotide seed region in the        sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin        inaccessibility decreases dCas9 binding to other sites with        matching seed sequences; thus 70% of off-target sites are        associated with genes. The authors showed that targeted        sequencing of 295 dCas9 binding sites in mESCs transfected with        catalytically active Cas9 identified only one site mutated above        background levels. The authors proposed a two-state model for        Cas9 binding and cleavage, in which a seed match triggers        binding but extensive pairing with target DNA is required for        cleavage.    -   Platt et al. established a Cre-dependent Cas9 knockin mouse. The        authors demonstrated in vivo as well as ex vivo genome editing        using adeno-associated virus (AAV)-, lentivirus-, or        particle-mediated delivery of guide RNA in neurons, immune        cells, and endothelial cells.    -   Hsu et al. (2014) is a review article that discusses generally        CRISPR-Cas9 history from yogurt to genome editing, including        genetic screening of cells.    -   Wang et al. (2014) relates to a pooled, loss-of-function genetic        screening approach suitable for both positive and negative        selection that uses a genome-scale lentiviral single guide RNA        (sgRNA) library.    -   Doench et al. created a pool of sgRNAs, tiling across all        possible target sites of a panel of six endogenous mouse and        three endogenous human genes and quantitatively assessed their        ability to produce null alleles of their target gene by antibody        staining and flow cytometry. The authors showed that        optimization of the PAM improved activity and also provided an        on-line tool for designing sgRNAs.    -   Swiech et al. demonstrate that AAV-mediated SpCas9 genome        editing can enable reverse genetic studies of gene function in        the brain.    -   Konermann et al. (2015) discusses the ability to attach multiple        effector domains, e.g., transcriptional activator, functional        and epigenomic regulators at appropriate positions on the guide        such as stem or tetraloop with and without linkers.    -   Zetsche et al. demonstrates that the Cas9 enzyme can be split        into two and hence the assembly of Cas9 for activation can be        controlled.    -   Chen et al. relates to multiplex screening by demonstrating that        a genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes        regulating lung metastasis.    -   Ran et al. (2015) relates to SaCas9 and its ability to edit        genomes and demonstrates that one cannot extrapolate from        biochemical assays.    -   Shalem et al. (2015) described ways in which catalytically        inactive Cas9 (dCas9) fusions are used to synthetically repress        (CRISPRi) or activate (CRISPRa) expression, showing. advances        using Cas9 for genome-scale screens, including arrayed and        pooled screens, knockout approaches that inactivate genomic loci        and strategies that modulate transcriptional activity.    -   Xu et al. (2015) assessed the DNA sequence features that        contribute to single guide RNA (sgRNA) efficiency in        CRISPR-based screens. The authors explored efficiency of        CRISPR-Cas9 knockout and nucleotide preference at the cleavage        site. The authors also found that the sequence preference for        CRISPRi/a is substantially different from that for CRISPR-Cas9        knockout.    -   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9        libraries into dendritic cells (DCs) to identify genes that        control the induction of tumor necrosis factor (Tnf) by        bacterial lipopolysaccharide (LPS). Known regulators of Tlr4        signaling and previously unknown candidates were identified and        classified into three functional modules with distinct effects        on the canonical responses to LPS.    -   Ramanan et al(2015) demonstrated cleavage of viral episomal DNA        (cccDNA) in infected cells. The HBV genome exists in the nuclei        of infected hepatocytes as a 3.2 kb double-stranded episomal DNA        species called covalently closed circular DNA (cccDNA), which is        a key component in the HBV life cycle whose replication is not        inhibited by current therapies. The authors showed that sgRNAs        specifically targeting highly conserved regions of HBV robustly        suppresses viral replication and depleted cccDNA.    -   Nishimasu et al. (2015) reported the crystal structures of        SaCas9 in complex with a single guide RNA (sgRNA) and its        double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and        the 5′-TTGGGT-3′ PAM. A structural comparison of SaCas9 with        SpCas9 highlighted both structural conservation and divergence,        explaining their distinct PAM specificities and orthologous        sgRNA recognition.    -   Canver et al. (2015) demonstrated a CRISPR-Cas9-based functional        investigation of non-coding genomic elements. The authors we        developed pooled CRISPR-Cas9 guide RNA libraries to perform in        situ saturating mutagenesis of the human and mouse BCL11A        enhancers which revealed critical features of the enhancers.    -   Zetsche et al. (2015) reported characterization of Cpf1, a class        2 CRISPR nuclease from Francisella novicida U112 having features        distinct from Cas9. Cpf1 is a single RNA-guided endonuclease        lacking tracrRNA, utilizes a T-rich protospacer-adjacent motif,        and cleaves DNA via a staggered DNA double-stranded break.    -   Shmakov et al. (2015) reported three distinct Class 2 CRISPR-Cas        systems. Two system CRISPR enzymes (C2c1 and C2c3) contain        RuvC-like endonuclease domains distantly related to Cpf1. Unlike        Cpf1, C2c1 depends on both crRNA and tracrRNA for DNA cleavage.        The third enzyme (C2c2) contains two predicted HEPN RNase        domains and is tracrRNA independent.    -   Slaymaker et al (2016) reported the use of structure-guided        protein engineering to improve the specificity of Streptococcus        pyogenes Cas9 (SpCas9). The authors developed “enhanced        specificity” SpCas9 (eSpCas9) variants which maintained robust        on-target cleavage with reduced off-target effects.

The methods and tools provided herein are may be designed for use withor Cas13, a type II nuclease that does not make use of tracrRNA.Orthologs of Cas13 have been identified in different bacterial speciesas described herein. Further type II nucleases with similar propertiescan be identified using methods described in the art (Shmakov et al.2015, 60:385-397; Abudayeh et al. 2016, Science, 5; 353(6299)). Inparticular embodiments, such methods for identifying novel CRISPReffector proteins may comprise the steps of selecting sequences from thedatabase encoding a seed which identifies the presence of a CRISPR Caslocus, identifying loci located within 10 kb of the seed comprising OpenReading Frames (ORFs) in the selected sequences, selecting therefromloci comprising ORFs of which only a single ORF encodes a novel CRISPReffector having greater than 700 amino acids and no more than 90%homology to a known CRISPR effector. In particular embodiments, the seedis a protein that is common to the CRISPR-Cas system, such as Cas1. Infurther embodiments, the CRISPR array is used as a seed to identify neweffector proteins.

Also, “Dimeric CRISPR RNA-guided FokI nucleases for highly specificgenome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter,Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin,Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77(2014), relates to dimeric RNA-guided FokI Nucleases that recognizeextended sequences and can edit endogenous genes with high efficienciesin human cells.

With respect to general information on CRISPR/Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, and making and using thereof,including as to amounts and formulations, as well asCRISPR-Cas-expressing eukaryotic cells, CRISPR-Cas expressingeukaryotes, such as a mouse, reference is made to: U.S. Pat. Nos.8,999,641, 8,993,233, 8,697,359, 8,771,945, 8,795,965, 8,865,406,8,871,445, 8,889,356, 8,889,418, 8,895,308, 8,906,616, 8,932,814, and8,945,839; US Patent Publications US 2014-0310830 (U.S. application Ser.No. 14/105,031), US 2014-0287938 A1 (U.S. application Ser. No.14/213,991), US 2014-0273234 A1 (U.S. application Ser. No. 14/293,674),US2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US2014-0273231 (U.S. application Ser. No. 14/259,420), US 2014-0256046 A1(U.S. application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S.application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. applicationSer. No. 14/222,930), US 2014-0242699 A1 (U.S. application Ser. No.14/183,512), US 2014-0242664 A1 (U.S. application Ser. No. 14/104,990),US 2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US2014-0227787 A1 (U.S. application Ser. No. 14/256,912), US 2014-0189896A1 (U.S. application Ser. No. 14/105,035), US 2014-0186958 (U.S.application Ser. No. 14/105,017), US 2014-0186919 A1 (U.S. applicationSer. No. 14/104,977), US 2014-0186843 A1 (U.S. application Ser. No.14/104,900), US 2014-0179770 A1 (U.S. application Ser. No. 14/104,837)and US 2014-0179006 A1 (U.S. application Ser. No. 14/183,486), US2014-0170753 (U.S. application Ser. No. 14/183,429); US 2015-0184139(U.S. application Ser. No. 14/324,960); 14/054,414 European PatentApplications EP 2 771 468 (EP13818570.7), EP 2 764 103 (EP13824232.6),and EP 2 784 162 (EP14170383.5); and PCT Patent PublicationsWO2014/093661 (PCT/US2013/074743), WO2014/093694 (PCT/US2013/074790),WO2014/093595 (PCT/US2013/074611), WO2014/093718 (PCT/US2013/074825),WO2014/093709 (PCT/US2013/074812), WO2014/093622 (PCT/US2013/074667),WO2014/093635 (PCT/US2013/074691), WO2014/093655 (PCT/US2013/074736),WO2014/093712 (PCT/US2013/074819), WO2014/093701 (PCT/US2013/074800),WO2014/018423 (PCT/US2013/051418), WO2014/204723 (PCT/US2014/041790),WO2014/204724 (PCT/US2014/041800), WO2014/204725 (PCT/US2014/041803),WO2014/204726 (PCT/US2014/041804), WO2014/204727 (PCT/US2014/041806),WO2014/204728 (PCT/US2014/041808), WO2014/204729 (PCT/US2014/041809),WO2015/089351 (PCT/US2014/069897), WO2015/089354 (PCT/US2014/069902),WO2015/089364 (PCT/US2014/069925), WO2015/089427 (PCT/US2014/070068),WO2015/089462 (PCT/US2014/070127), WO2015/089419 (PCT/US2014/070057),WO2015/089465 (PCT/US2014/070135), WO2015/089486 (PCT/US2014/070175),WO2015/058052 (PCT/US2014/061077), WO2015/070083 (PCT/US2014/064663),WO2015/089354 (PCT/US2014/069902), WO2015/089351 (PCT/US2014/069897),WO2015/089364 (PCT/US2014/069925), WO2015/089427 (PCT/US2014/070068),WO2015/089473 (PCT/US2014/070152), WO2015/089486 (PCT/US2014/070175),WO2016/049258 (PCT/US2015/051830), WO2016/094867 (PCT/US2015/065385),WO2016/094872 (PCT/US2015/065393), WO2016/094874 (PCT/US2015/065396),WO2016/106244 (PCT/US2015/067177).

Mention is also made of U.S. application 62/180,709, 17 Jun. 2015,PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,455, filed, 12Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708,24 Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. applications62/091,462, 12 Dec. 2014, 62/096,324, 23 Dec. 2014, 62/180,681, 17 Jun.2015, and 62/237,496, 5 Oct. 2015, DEAD GUIDES FOR CRISPR TRANSCRIPTIONFACTORS; U.S. application 62/091,456, 12 Dec. 2014 and 62/180,692, 17Jun. 2015, ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS;U.S. application 62/091,461, 12 Dec. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOMEEDITING AS TO HEMATOPOETIC STEM CELLS (HSCs); U.S. application62/094,903, 19 Dec. 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRANDBREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURESEQUENCING; U.S. application 62/096,761, 24 Dec. 2014, ENGINEERING OFSYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCEMANIPULATION; U.S. application 62/098,059, 30 Dec. 2014, 62/181,641, 18Jun. 2015, and 62/181,667, 18 Jun. 2015, RNA-TARGETING SYSTEM; U.S.application 62/096,656, 24 Dec. 2014 and 62/181,151, 17 Jun. 2015,CRISPR HAVING OR ASSOCIATED WITH DESTABILIZATION DOMAINS; U.S.application 62/096,697, 24 Dec. 2014, CRISPR HAVING OR ASSOCIATED WITHAAV; U.S. application 62/098,158, 30 Dec. 2014, ENGINEERED CRISPRCOMPLEX INSERTIONAL TARGETING SYSTEMS; U.S. application 62/151,052, 22Apr. 2015, CELLULAR TARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; U.S.application 62/054,490, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS; U.S.application 61/939,154, 12 Feb. 2014, SYSTEMS, METHODS AND COMPOSITIONSFOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;U.S. application 62/055,484, 25 Sep. 2014, SYSTEMS, METHODS ANDCOMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONALCRISPR-CAS SYSTEMS; U.S. application 62/087,537, 4 Dec. 2014, SYSTEMS,METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/054,651, 24 Sep.2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCERMUTATIONS IN VIVO; U.S. application 62/067,886, 23 Oct. 2014, DELIVERY,USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS ANDCOMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS INVIVO; U.S. applications 62/054,675, 24 Sep. 2014 and 62/181,002, 17 Jun.2015, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS IN NEURONAL CELLS/TISSUES; U.S. application62/054,528, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN IMMUNE DISEASES OR DISORDERS;U.S. application 62/055,454, 25 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING CELL PENETRATION PEPTIDES (CPP); U.S.application 62/055,460, 25 Sep. 2014, MULTIFUNCTIONAL-CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; U.S.application 62/087,475, 4 Dec. 2014 and 62/181,690, 18 Jun. 2015,FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/055,487, 25 Sep. 2014, FUNCTIONAL SCREENING WITHOPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/087,546, 4Dec. 2014 and 62/181,687, 18 Jun. 2015, MULTIFUNCTIONAL CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; and U.S.application 62/098,285, 30 Dec. 2014, CRISPR MEDIATED IN VIVO MODELINGAND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

Mention is made of U.S. applications 62/181,659, 18 Jun. 2015 and62/207,318, 19 Aug. 2015, ENGINEERING AND OPTIMIZATION OF SYSTEMS,METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FORSEQUENCE MANIPULATION. Mention is made of U.S. applications 62/181,663,18 Jun. 2015 and 62/245,264, 22 Oct. 2015, NOVEL CRISPR ENZYMES ANDSYSTEMS, U.S. applications 62/181,675, 18 Jun. 2015, 62/285,349, 22 Oct.2015, 62/296,522, 17 Feb. 2016, and 62/320,231, 8 Apr. 2016, NOVELCRISPR ENZYMES AND SYSTEMS, U.S. application 62/232,067, 24 Sep. 2015,U.S. application Ser. No. 14/975,085, 18 Dec. 2015, European applicationNo. 16150428.7, U.S. application 62/205,733, 16 Aug. 2015, U.S.application 62/201,542, 5 Aug. 2015, U.S. application 62/193,507, 16Jul. 2015, and U.S. application 62/181,739, 18 Jun. 2015, each entitledNOVEL CRISPR ENZYMES AND SYSTEMS and of U.S. application 62/245,270, 22Oct. 2015, NOVEL CRISPR ENZYMES AND SYSTEMS. Mention is also made ofU.S. application 61/939,256, 12 Feb. 2014, and WO 2015/089473(PCT/US2014/070152), 12 Dec. 2014, each entitled ENGINEERING OF SYSTEMS,METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURES FORSEQUENCE MANIPULATION. Mention is also made of PCT/US2015/045504, 15Aug. 2015, U.S. application 62/180,699, 17 Jun. 2015, and U.S.application 62/038,358, 17 Aug. 2014, each entitled GENOME EDITING USINGCAS9 NICKASES.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appln citeddocuments”) and all documents cited or referenced in the appln citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appln cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

2. Tale Systems

As disclosed herein editing can be made by way of the transcriptionactivator-like effector nucleases (TALENs) system. Transcriptionactivator-like effectors (TALEs) can be engineered to bind practicallyany desired DNA sequence. Exemplary methods of genome editing using theTALEN system can be found for example in Cermak T. Doyle EL. ChristianM. Wang L. Zhang Y. Schmidt C, et al. Efficient design and assembly ofcustom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic Acids Res. 2011; 39:e82; Zhang F. Cong L. Lodato S. Kosuri S.Church GM. Arlotta P Efficient construction of sequence-specific TALeffectors for modulating mammalian transcription. Nat Biotechnol. 2011;29:149-153 and U.S. Pat. Nos. 8,450,471, 8,440,431 and 8,440,432, all ofwhich are specifically incorporated by reference.

In advantageous embodiments of the invention, the methods providedherein use isolated, non-naturally occurring, recombinant or engineeredDNA binding proteins that comprise TALE monomers as a part of theirorganizational structure that enable the targeting of nucleic acidsequences with improved efficiency and expanded specificity.

Naturally occurring TALEs or “wild type TALEs” are nucleic acid bindingproteins secreted by numerous species of proteobacteria. TALEpolypeptides contain a nucleic acid binding domain composed of tandemrepeats of highly conserved monomer polypeptides that are predominantly33, 34 or 35 amino acids in length and that differ from each othermainly in amino acid positions 12 and 13. In advantageous embodimentsthe nucleic acid is DNA. As used herein, the term “polypeptidemonomers”, or “TALE monomers” will be used to refer to the highlyconserved repetitive polypeptide sequences within the TALE nucleic acidbinding domain and the term “repeat variable di-residues” or “RVD” willbe used to refer to the highly variable amino acids at positions 12 and13 of the polypeptide monomers. As provided throughout the disclosure,the amino acid residues of the RVD are depicted using the IUPAC singleletter code for amino acids. A general representation of a TALE monomerwhich is comprised within the DNA binding domain isX1-11-(X12X13)-X14-33 or 34 or 35, where the subscript indicates theamino acid position and X represents any amino acid. X12X13 indicate theRVDs. In some polypeptide monomers, the variable amino acid at position13 is missing or absent and in such polypeptide monomers, the RVDconsists of a single amino acid. In such cases the RVD may bealternatively represented as X*, where X represents X12 and (*)indicates that X13 is absent. The DNA binding domain comprises severalrepeats of TALE monomers and this may be represented as(X1-11-(X12X13)-X14-33 or 34 or 35)z, where in an advantageousembodiment, z is at least 5 to 40. In a further advantageous embodiment,z is at least 10 to 26.

The TALE monomers have a nucleotide binding affinity that is determinedby the identity of the amino acids in its RVD. For example, polypeptidemonomers with an RVD of NI preferentially bind to adenine (A),polypeptide monomers with an RVD of NG preferentially bind to thymine(T), polypeptide monomers with an RVD of HD preferentially bind tocytosine (C) and polypeptide monomers with an RVD of NN preferentiallybind to both adenine (A) and guanine (G). In yet another embodiment ofthe invention, polypeptide monomers with an RVD of IG preferentiallybind to T. Thus, the number and order of the polypeptide monomer repeatsin the nucleic acid binding domain of a TALE determines its nucleic acidtarget specificity. In still further embodiments of the invention,polypeptide monomers with an RVD of NS recognize all four base pairs andmay bind to A, T, G or C. The structure and function of TALEs is furtherdescribed in, for example, Moscou et al., Science 326:1501 (2009); Bochet al., Science 326:1509-1512 (2009); and Zhang et al., NatureBiotechnology 29:149-153 (2011), each of which is incorporated byreference in its entirety.

The TALE polypeptides used in methods of the invention are isolated,non-naturally occurring, recombinant or engineered nucleic acid-bindingproteins that have nucleic acid or DNA binding regions containingpolypeptide monomer repeats that are designed to target specific nucleicacid sequences.

As described herein, polypeptide monomers having an RVD of HN or NHpreferentially bind to guanine and thereby allow the generation of TALEpolypeptides with high binding specificity for guanine containing targetnucleic acid sequences. In a preferred embodiment of the invention,polypeptide monomers having RVDs RN, NN, NK, SN, NH, KN, HN, NQ, HH, RG,KH, RH and SS preferentially bind to guanine. In a much moreadvantageous embodiment of the invention, polypeptide monomers havingRVDs RN, NK, NQ, HH, KH, RH, SS and SN preferentially bind to guanineand thereby allow the generation of TALE polypeptides with high bindingspecificity for guanine containing target nucleic acid sequences. In aneven more advantageous embodiment of the invention, polypeptide monomershaving RVDs HH, KH, NH, NK, NQ, RH, RN and SS preferentially bind toguanine and thereby allow the generation of TALE polypeptides with highbinding specificity for guanine containing target nucleic acidsequences. In a further advantageous embodiment, the RVDs that have highbinding specificity for guanine are RN, NH RH and KH. Furthermore,polypeptide monomers having an RVD of NV preferentially bind to adenineand guanine. In more preferred embodiments of the invention, polypeptidemonomers having RVDs of H*, HA, KA, N*, NA, NC, NS, RA, and S* bind toadenine, guanine, cytosine and thymine with comparable affinity.

The predetermined N-terminal to C-terminal order of the one or morepolypeptide monomers of the nucleic acid or DNA binding domaindetermines the corresponding predetermined target nucleic acid sequenceto which the TALE polypeptides will bind. As used herein the polypeptidemonomers and at least one or more half polypeptide monomers are“specifically ordered to target” the genomic locus or gene of interest.In plant genomes, the natural TALE-binding sites always begin with athymine (T), which may be specified by a cryptic signal within thenon-repetitive N-terminus of the TALE polypeptide; in some cases thisregion may be referred to as repeat 0. In animal genomes, TALE bindingsites do not necessarily have to begin with a thymine (T) and TALEpolypeptides may target DNA sequences that begin with T, A, G or C. Thetandem repeat of TALE monomers always ends with a half-length repeat ora stretch of sequence that may share identity with only the first 20amino acids of a repetitive full length TALE monomer and this halfrepeat may be referred to as a half-monomer (FIG. 8), which is includedin the term “TALE monomer”. Therefore, it follows that the length of thenucleic acid or DNA being targeted is equal to the number of fullpolypeptide monomers plus two.

As described in Zhang et al., Nature Biotechnology 29:149-153 (2011),TALE polypeptide binding efficiency may be increased by including aminoacid sequences from the “capping regions” that are directly N-terminalor C-terminal of the DNA binding region of naturally occurring TALEsinto the engineered TALEs at positions N-terminal or C-terminal of theengineered TALE DNA binding region. Thus, in certain embodiments, theTALE polypeptides described herein further comprise an N-terminalcapping region and/or a C-terminal capping region.

An exemplary amino acid sequence of a N-terminal capping region is:

(SEQ ID NO: 3)M D P I R S R T P S P A R E L L S G P Q P D G V Q P T A D R G V S PP A G G P L D G L P A R R T M S R T R L P S P P A P S P A F S A D SF S D L L R Q F D P S L F N T S L F D S L P P F G A H H T E A A T GE W D E V Q S G L R A A D A P P P T M R V A V T A A R P P R A K P AP R R R A A Q P S D A S P A A Q V D L R T L G Y S Q Q Q Q E K I K PK V R S T V A Q H H E A L V G H G F T H A H I V A L S Q H P A A L GT V A V K Y Q D M I A A L P E A T H E A I V G V G K Q W S G A R A LE A L L T V A G E L R G P P L Q L D T G Q L L K I A K R G G V T A VE A V H A W R N A L T G A P L N

An exemplary amino acid sequence of a C-terminal capping region is:

(SEQ ID NO: 4)R P A L E S I V A Q L S R P D P A L A A L T N D H L V A L A C L GG R P A L D A V K K G L P H A P A L I K R T N R R I P E R T S H RV A D H A Q V V R V L G F F Q C H S H P A Q A F D D A M T Q F G MS R H G L L Q L F R R V G V T E L E A R S G T L P P A S Q R W D RI L Q A S G M K R A K P S P T S T Q T P D Q A S L H A F A D S L ER D L D A P S P M H E G D Q T R A S

As used herein the predetermined “N-terminus” to “C terminus”orientation of the N-terminal capping region, the DNA binding domaincomprising the repeat TALE monomers and the C-terminal capping regionprovide structural basis for the organization of different domains inthe d-TALEs or polypeptides of the invention.

The entire N-terminal and/or C-terminal capping regions are notnecessary to enhance the binding activity of the DNA binding region.Therefore, in certain embodiments, fragments of the N-terminal and/orC-terminal capping regions are included in the TALE polypeptidesdescribed herein.

In certain embodiments, the TALE polypeptides described herein contain aN-terminal capping region fragment that included at least 10, 20, 30,40, 50, 54, 60, 70, 80, 87, 90, 94, 100, 102, 110, 117, 120, 130, 140,147, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or 270amino acids of an N-terminal capping region. In certain embodiments, theN-terminal capping region fragment amino acids are of the C-terminus(the DNA-binding region proximal end) of an N-terminal capping region.As described in Zhang et al., Nature Biotechnology 29:149-153 (2011),N-terminal capping region fragments that include the C-terminal 240amino acids enhance binding activity equal to the full length cappingregion, while fragments that include the C-terminal 147 amino acidsretain greater than 80% of the efficacy of the full length cappingregion, and fragments that include the C-terminal 117 amino acids retaingreater than 50% of the activity of the full-length capping region.

In some embodiments, the TALE polypeptides described herein contain aC-terminal capping region fragment that included at least 6, 10, 20, 30,37, 40, 50, 60, 68, 70, 80, 90, 100, 110, 120, 127, 130, 140, 150, 155,160, 170, 180 amino acids of a C-terminal capping region. In certainembodiments, the C-terminal capping region fragment amino acids are ofthe N-terminus (the DNA-binding region proximal end) of a C-terminalcapping region. As described in Zhang et al., Nature Biotechnology29:149-153 (2011), C-terminal capping region fragments that include theC-terminal 68 amino acids enhance binding activity equal to the fulllength capping region, while fragments that include the C-terminal 20amino acids retain greater than 50% of the efficacy of the full lengthcapping region.

In certain embodiments, the capping regions of the TALE polypeptidesdescribed herein do not need to have identical sequences to the cappingregion sequences provided herein. Thus, in some embodiments, the cappingregion of the TALE polypeptides described herein have sequences that areat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical or share identity to the capping region aminoacid sequences provided herein. Sequence identity is related to sequencehomology. Homology comparisons may be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesecommercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences. In some preferred embodiments, the capping region of the TALEpolypeptides described herein have sequences that are at least 95%identical or share identity to the capping region amino acid sequencesprovided herein.

Sequence homologies may be generated by any of a number of computerprograms known in the art, which include but are not limited to BLAST orFASTA. Suitable computer program for carrying out alignments like theGCG Wisconsin Bestfit package may also be used. Once the software hasproduced an optimal alignment, it is possible to calculate % homology,preferably % sequence identity. The software typically does this as partof the sequence comparison and generates a numerical result.

In advantageous embodiments described herein, the TALE polypeptides ofthe invention include a nucleic acid binding domain linked to the one ormore effector domains. The terms “effector domain” or “regulatory andfunctional domain” refer to a polypeptide sequence that has an activityother than binding to the nucleic acid sequence recognized by thenucleic acid binding domain. By combining a nucleic acid binding domainwith one or more effector domains, the polypeptides of the invention maybe used to target the one or more functions or activities mediated bythe effector domain to a particular target DNA sequence to which thenucleic acid binding domain specifically binds.

In some embodiments of the TALE polypeptides described herein, theactivity mediated by the effector domain is a biological activity. Forexample, in some embodiments the effector domain is a transcriptionalinhibitor (i.e., a repressor domain), such as an mSin interaction domain(SID). SID4X domain or a Kruppel-associated box (KRAB) or fragments ofthe KRAB domain. In some embodiments the effector domain is an enhancerof transcription (i.e. an activation domain), such as the VP16, VP64 orp65 activation domain. In some embodiments, the nucleic acid binding islinked, for example, with an effector domain that includes but is notlimited to a transposase, integrase, recombinase, resolvase, invertase,protease, DNA methyltransferase, DNA demethylase, histone acetylase,histone deacetylase, nuclease, transcriptional repressor,transcriptional activator, transcription factor recruiting, proteinnuclear-localization signal or cellular uptake signal.

In some embodiments, the effector domain is a protein domain whichexhibits activities which include but are not limited to transposaseactivity, integrase activity, recombinase activity, resolvase activity,invertase activity, protease activity, DNA methyltransferase activity,DNA demethylase activity, histone acetylase activity, histonedeacetylase activity, nuclease activity, nuclear-localization signalingactivity, transcriptional repressor activity, transcriptional activatoractivity, transcription factor recruiting activity, or cellular uptakesignaling activity. Other preferred embodiments of the invention mayinclude any combination the activities described herein.

3. ZN-Finger Nucleases

Other preferred tools for genome editing for use in the context of thisinvention include zinc finger systems and TALE systems. One type ofprogrammable DNA-binding domain is provided by artificial zinc-finger(ZF) technology, which involves arrays of ZF modules to target newDNA-binding sites in the genome. Each finger module in a ZF arraytargets three DNA bases. A customized array of individual zinc fingerdomains is assembled into a ZF protein (ZFP).

ZFPs can comprise a functional domain. The first synthetic zinc fingernucleases (ZFNs) were developed by fusing a ZF protein to the catalyticdomain of the Type IIS restriction enzyme FokI. (Kim, Y. G. et al.,1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. U.S.A.91, 883-887; Kim, Y. G. et al., 1996, Hybrid restriction enzymes: zincfinger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. U.S.A.93, 1156-1160). Increased cleavage specificity can be attained withdecreased off target activity by use of paired ZFN heterodimers, eachtargeting different nucleotide sequences separated by a short spacer.(Doyon, Y. et al., 2011, Enhancing zinc-finger-nuclease activity withimproved obligate heterodimeric architectures. Nat. Methods 8, 74-79).ZFPs can also be designed as transcription activators and repressors andhave been used to target many genes in a wide variety of organisms.Exemplary methods of genome editing using ZFNs can be found for examplein U.S. Pat. Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978,6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719,7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626,all of which are specifically incorporated by reference.

4. Meganucleases

As disclosed herein editing can be made by way of meganucleases, whichare endodeoxyribonucleases characterized by a large recognition site(double-stranded DNA sequences of 12 to 40 base pairs). Exemplary methodfor using meganucleases can be found in U.S. Pat. Nos. 8,163,514;8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,369; and 8,129,134,which are specifically incorporated by reference.

Delivery

The programmable nucleic acid modifying agents and other modulatingagents, or components thereof, or nucleic acid molecules thereof(including, for instance HDR template), or nucleic acid moleculesencoding or providing components thereof, may be delivered by a deliverysystem herein described.

Viral Delivery

Vector delivery, e.g., plasmid, viral delivery: the chromatin 3Dstructure modulating agents, can be delivered using any suitable vector,e.g., plasmid or viral vectors, such as adeno associated virus (AAV),lentivirus, adenovirus or other viral vector types, or combinationsthereof. In some embodiments, the vector, e.g., plasmid or viral vectoris delivered to the tissue of interest by, for example, an intramuscularinjection, while other times the delivery is via intravenous,transdermal, intranasal, oral, mucosal, or other delivery methods. Suchdelivery may be either via a single dose, or multiple doses. One skilledin the art understands that the actual dosage to be delivered herein mayvary greatly depending upon a variety of factors, such as the vectorchoice, the target cell, organism, or tissue, the general condition ofthe subject to be treated, the degree of transformation/modificationsought, the administration route, the administration mode, the type oftransformation/modification sought, etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

Compositions comprising a Cas effector module, complex or systemcomprising multiple guide RNAs, preferably tandemly arranged, or thepolynucleotide or vector encoding or comprising said Cas effectormodule, complex or system comprising multiple guide RNAs, preferablytandemly arranged, for use in the methods of treatment as defined hereinelsewhere are also provided. A kit of parts may be provided includingsuch compositions. Use of said composition in the manufacture of amedicament for such methods of treatment are also provided. Use of a Caseffector module CRISPR system in screening is also provided by thepresent invention, e.g., gain of function screens. Cells which areartificially forced to overexpress a gene are be able to down regulatethe gene over time (re-establishing equilibrium) e.g. by negativefeedback loops. By the time the screen starts the unregulated gene mightbe reduced again. Using an inducible Cas effector module activatorallows one to induce transcription right before the screen and thereforeminimizes the chance of false negative hits. Accordingly, by use of theinstant invention in screening, e.g., gain of function screens, thechance of false negative results may be minimized.

In another aspect, the invention provides an engineered, non-naturallyoccurring vector system comprising one or more vectors comprising afirst regulatory element operably linked to the multiple Cas effectormodule CRISPR system guide RNAs that each specifically target a DNAmolecule encoding a gene product and a second regulatory elementoperably linked coding for a CRISPR protein. Both regulatory elementsmay be located on the same vector or on different vectors of the system.The multiple guide RNAs target the multiple DNA molecules encoding themultiple gene products in a cell and the CRISPR protein may cleave themultiple DNA molecules encoding the gene products (it may cleave one orboth strands or have substantially no nuclease activity), wherebyexpression of the multiple gene products is altered; and, wherein theCRISPR protein and the multiple guide RNAs do not naturally occurtogether. In a preferred embodiment the CRISPR protein is a Cas effectormodule, optionally codon optimized for expression in a eukaryotic cell.In a preferred embodiment the eukaryotic cell is a mammalian cell, aplant cell or a yeast cell and in a more preferred embodiment themammalian cell is a human cell. In a further embodiment of theinvention, the expression of each of the multiple gene products isaltered, preferably decreased.

In one aspect, the invention provides a vector system comprising one ormore vectors. In some embodiments, the system comprises: (a) a firstregulatory element operably linked to a direct repeat sequence and oneor more insertion sites for inserting one or more guide sequences up- ordownstream (whichever applicable) of the direct repeat sequence, whereinwhen expressed, the one or more guide sequence(s) direct(s)sequence-specific binding of the CRISPR complex to the one or moretarget sequence(s) in a eukaryotic cell, wherein the CRISPR complexcomprises a Cas effector module complexed with the one or more guidesequence(s) that is hybridized to the one or more target sequence(s);and (b) a second regulatory element operably linked to an enzyme-codingsequence encoding said Cas effector module, preferably comprising atleast one nuclear localization sequence and/or at least one NES; whereincomponents (a) and (b) are located on the same or different vectors ofthe system. In some embodiments, component (a) further comprises two ormore guide sequences operably linked to the first regulatory element,wherein when expressed, each of the two or more guide sequences directsequence specific binding of a CRISPR complex to a different targetsequence in a eukaryotic cell. In some embodiments, the CRISPR complexcomprises one or more nuclear localization sequences and/or one or moreNES of sufficient strength to drive accumulation of said CRISPR complexin a detectable amount in or out of the nucleus of a eukaryotic cell. Insome embodiments, the first regulatory element is a polymerase IIIpromoter. In some embodiments, the second regulatory element is apolymerase II promoter. In some embodiments, each of the guide sequencesis at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, orbetween 16-25, or between 16-20 nucleotides in length.

Recombinant expression vectors can comprise the polynucleotides encodingthe Cas effector module, system or complex for use in multiple targetingas defined herein in a form suitable for expression of the nucleic acidin a host cell, which means that the recombinant expression vectorsinclude one or more regulatory elements, which may be selected on thebasis of the host cells to be used for expression, that isoperatively-linked to the nucleic acid sequence to be expressed. Withina recombinant expression vector, “operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatoryelement(s) in a manner that allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell).

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors comprising the polynucleotidesencoding the Cas effector module, system or complex for use in multipletargeting as defined herein. In some embodiments, a cell is transfectedas it naturally occurs in a subject. In some embodiments, a cell that istransfected is taken from a subject. In some embodiments, the cell isderived from cells taken from a subject, such as a cell line. A widevariety of cell lines for tissue culture are known in the art andexemplified herein elsewhere. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors comprising the polynucleotidesencoding the Cas effector module, system or complex for use in multipletargeting as defined herein is used to establish a new cell linecomprising one or more vector-derived sequences. In some embodiments, acell transiently transfected with the components of a Cas effectormodule. system or complex for use in multiple targeting as describedherein (such as by transient transfection of one or more vectors, ortransfection with RNA), and modified through the activity of a Caseffector module, system or complex, is used to establish a new cell linecomprising cells containing the modification but lacking any otherexogenous sequence. In some embodiments, cells transiently ornon-transiently transfected with one or more vectors comprising thepolynucleotides encoding Cas effector module, system or complex for usein multiple targeting as defined herein, or cell lines derived from suchcells are used in assessing one or more test compounds.

The term “regulatory element” is as defined herein elsewhere.

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

In one aspect, the invention provides a eukaryotic host cell comprising(a) a first regulatory element operably linked to a direct repeatsequence and one or more insertion sites for inserting one or more guideRNA sequences up- or downstream (whichever applicable) of the directrepeat sequence, wherein when expressed, the guide sequence(s) direct(s)sequence-specific binding of the CRISPR complex to the respective targetsequence(s) in a eukaryotic cell, wherein the CRISPR complex comprises aCas effector module complexed with the one or more guide sequence(s)that is hybridized to the respective target sequence(s); and/or (b) asecond regulatory element operably linked to an enzyme-coding sequenceencoding said Cas effector module comprising preferably at least onenuclear localization sequence and/or NES. In some embodiments, the hostcell comprises components (a) and (b). In some embodiments, component(a), component (b), or components (a) and (b) are stably integrated intoa genome of the host eukaryotic cell. In some embodiments, component (a)further comprises two or more guide sequences operably linked to thefirst regulatory element, and optionally separated by a direct repeat,wherein when expressed, each of the two or more guide sequences directsequence specific binding of a CRISPR complex to a different targetsequence in a eukaryotic cell. In some embodiments, the Cas effectormodule comprises one or more nuclear localization sequences and/ornuclear export sequences or NES of sufficient strength to driveaccumulation of said CRISPR enzyme in a detectable amount in and/or outof the nucleus of a eukaryotic cell.

Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

In certain aspects the invention involves vectors. A used herein, a“vector” is a tool that allows or facilitates the transfer of an entityfrom one environment to another. It is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. Generally, avector is capable of replication when associated with the proper controlelements. In general, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. Vectors include, but are not limited to, nucleic acidmolecules that are single-stranded, double-stranded, or partiallydouble-stranded; nucleic acid molecules that comprise one or more freeends, no free ends (e.g. circular); nucleic acid molecules that compriseDNA, RNA, or both; and other varieties of polynucleotides known in theart. One type of vector is a “plasmid,” which refers to a circulardouble stranded DNA loop into which additional DNA segments can beinserted, such as by standard molecular cloning techniques. Another typeof vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses(AAVs)). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g. bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

The vector(s) can include the regulatory element(s), e.g., promoter(s).The vector(s) can comprise Cas encoding sequences, and/or a single, butpossibly also can comprise at least 3 or 8 or 16 or 32 or 48 or 50 guideRNA(s) (e.g., sgRNAs) encoding sequences, such as 1-2, 1-3, 1-4 1-5,3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s)(e.g., sgRNAs). In a single vector there can be a promoter for each RNA(e.g., sgRNA), advantageously when there are up to about 16 RNA(s)(e.g., sgRNAs); and, when a single vector provides for more than 16RNA(s) (e.g., sgRNAs), one or more promoter(s) can drive expression ofmore than one of the RNA(s) (e.g., sgRNAs), e.g., when there are 32RNA(s) (e.g., sgRNAs), each promoter can drive expression of two RNA(s)(e.g., sgRNAs), and when there are 48 RNA(s) (e.g., sgRNAs), eachpromoter can drive expression of three RNA(s) (e.g., sgRNAs). By simplearithmetic and well established cloning protocols and the teachings inthis disclosure one skilled in the art can readily practice theinvention as to the RNA(s) (e.g., sgRNA(s) for a suitable exemplaryvector such as AAV, and a suitable promoter such as the U6 promoter,e.g., U6-sgRNAs. For example, the packaging limit of AAV is ˜4.7 kb. Thelength of a single U6-sgRNA (plus restriction sites for cloning) is 361bp. Therefore, the skilled person can readily fit about 12-16, e.g., 13U6-sgRNA cassettes in a single vector. This can be assembled by anysuitable means, such as a golden gate strategy used for TALE assembly(www.genome-engineering.org/taleffectors/). The skilled person can alsouse a tandem guide strategy to increase the number of U6-sgRNAs byapproximately 1.5 times, e.g., to increase from 12-16, e.g., 13 toapproximately 18-24, e.g., about 19 U6-sgRNAs. Therefore, one skilled inthe art can readily reach approximately 18-24, e.g., about 19promoter-RNAs, e.g., U6-sgRNAs in a single vector, e.g., an AAV vector.A further means for increasing the number of promoters and RNAs, e.g.,sgRNA(s) in a vector is to use a single promoter (e.g., U6) to expressan array of RNAs, e.g., sgRNAs separated by cleavable sequences. And aneven further means for increasing the number of promoter-RNAs, e.g.,sgRNAs in a vector, is to express an array of promoter-RNAs, e.g.,sgRNAs separated by cleavable sequences in the intron of a codingsequence or gene; and, in this instance it is advantageous to use apolymerase II promoter, which can have increased expression and enablethe transcription of long RNA in a tissue specific manner. (see, e.g.,nar.oxfordjournals.org/content/34/7/e53.short,www.nature.com/mt/journal/v16/n9/abs/mt2008144a.html). In anadvantageous embodiment, AAV may package U6 tandem sgRNA targeting up toabout 50 genes. Accordingly, from the knowledge in the art and theteachings in this disclosure the skilled person can readily make and usevector(s), e.g., a single vector, expressing multiple RNAs or guides orsgRNAs under the control or operatively or functionally linked to one ormore promoters-especially as to the numbers of RNAs or guides or sgRNAsdiscussed herein, without any undue experimentation.

The guide RNA(s), e.g., sgRNA(s) encoding sequences and/or Cas encodingsequences, can be functionally or operatively linked to regulatoryelement(s) and hence the regulatory element(s) drive expression. Thepromoter(s) can be constitutive promoter(s) and/or conditionalpromoter(s) and/or inducible promoter(s) and/or tissue specificpromoter(s). The promoter can be selected from the group consisting ofRNA polymerases, pol I, pol II, pol III, T7, U6, H1, retroviral Roussarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter,the SV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter. An advantageous promoter is the promoter is U6.

Aspects of the invention relate to bicistronic vectors for guide RNA and(optionally modified or mutated) Cas effector modules. Bicistronicexpression vectors for guide RNA and (optionally modified or mutated)CRISPR enzymes are preferred. In general and particularly in thisembodiment (optionally modified or mutated) CRISPR enzymes arepreferably driven by the CBh promoter. The RNA may preferably be drivenby a Pol III promoter, such as a U6 promoter. Ideally the two arecombined.

In some embodiments, a loop in the guide RNA is provided. This may be astem loop or a tetra loop. The loop is preferably GAAA, but it is notlimited to this sequence or indeed to being only 4 bp in length. Indeed,preferred loop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG.

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g. transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g. liver,pancreas), or particular cell types (e.g. lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g. 1, 2,3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g.1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters(e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.Examples of pol III promoters include, but are not limited to, U6 and H1promoters. Examples of pol II promoters include, but are not limited to,the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally withthe RSV enhancer), the cytomegalovirus (CMV) promoter (optionally withthe CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],the SV40 promoter, the dihydrofolate reductase promoter, the R-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter. Also encompassed by the term “regulatory element” are enhancerelements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR ofHTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer;and the intron sequence between exons 2 and 3 of rabbit P-globin (Proc.Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression desired, etc. A vectorcan be introduced into host cells to thereby produce transcripts,proteins, or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., clustered regularlyinterspersed short palindromic repeats (CRISPR) transcripts, proteins,enzymes, mutant forms thereof, fusion proteins thereof, etc.). Withregards to regulatory sequences, mention is made of U.S. patentapplication Ser. No. 10/491,026, the contents of which are incorporatedby reference herein in their entirety. With regards to promoters,mention is made of PCT publication WO 2011/028929 and U.S. applicationSer. No. 12/511,940, the contents of which are incorporated by referenceherein in their entirety.

Vectors can be designed for expression of CRISPR transcripts (e.g.nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g. amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amrann etal., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif (1990) 60-89). In some embodiments, a vector is a yeastexpression vector. Examples of vectors for expression in yeastSaccharomyces cerivisae include pYepSecl (Baldari, et al., 1987. EMBO J.6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943),pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,Calif). In some embodiments, a vector drives protein expression ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the a-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546). With regards to theseprokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No.6,750,059, the contents of which are incorporated by reference herein intheir entirety. Other embodiments of the invention may relate to the useof viral vectors, with regards to which mention is made of U.S. patentapplication Ser. No. 13/092,085, the contents of which are incorporatedby reference herein in their entirety. Tissue-specific regulatoryelements are known in the art and in this regard, mention is made ofU.S. Pat. No. 7,776,321, the contents of which are incorporated byreference herein in their entirety. In some embodiments, a regulatoryelement is operably linked to one or more elements of a CRISPR system soas to drive expression of the one or more elements of the CRISPR system.In general, CRISPRs (Clustered Regularly Interspaced Short PalindromicRepeats), also known as SPIDRs (SPacer Interspersed Direct Repeats),constitute a family of DNA loci that are usually specific to aparticular bacterial species. The CRISPR locus comprises a distinctclass of interspersed short sequence repeats (SSRs) that were recognizedin E. coli (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; andNakata et al., J. Bacteriol., 171:3553-3556 [1989]), and associatedgenes. Similar interspersed SSRs have been identified in Haloferaxmediterranei, Streptococcus pyogenes, Anabaena, and Mycobacteriumtuberculosis (See, Groenen et al., Mol. Microbiol., 10:1057-1065 [1993];Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999]; Masepohl et al.,Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica et al., Mol.Microbiol., 17:85-93 [1995]). The CRISPR loci typically differ fromother SSRs by the structure of the repeats, which have been termed shortregularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol.,6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]).In general, the repeats are short elements that occur in clusters thatare regularly spaced by unique intervening sequences with asubstantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

Typically, in the context of an endogenous nucleic acid-targetingsystem, formation of a nucleic acid-targeting complex (comprising aguide RNA hybridized to a target sequence and complexed with one or morenucleic acid-targeting effector modules) results in cleavage of one orboth RNA strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, or more base pairs from) the target sequence. In someembodiments, one or more vectors driving expression of one or moreelements of a nucleic acid-targeting system are introduced into a hostcell such that expression of the elements of the nucleic acid-targetingsystem direct formation of a nucleic acid-targeting complex at one ormore target sites. For example, a nucleic acid-targeting effector moduleand a guide RNA could each be operably linked to separate regulatoryelements on separate vectors. Alternatively, two or more of the elementsexpressed from the same or different regulatory elements, may becombined in a single vector, with one or more additional vectorsproviding any components of the nucleic acid-targeting system notincluded in the first vector. nucleic acid-targeting system elementsthat are combined in a single vector may be arranged in any suitableorientation, such as one element located 5′ with respect to (“upstream”of) or 3′ with respect to (“downstream” of) a second element. The codingsequence of one element may be located on the same or opposite strand ofthe coding sequence of a second element, and oriented in the same oropposite direction. In some embodiments, a single promoter drivesexpression of a transcript encoding a nucleic acid-targeting effectormodule and a guide RNA embedded within one or more intron sequences(e.g. each in a different intron, two or more in at least one intron, orall in a single intron). In some embodiments, the nucleic acid-targetingeffector module and guide RNA are operably linked to and expressed fromthe same promoter.

Ways to package inventive Cpf1 coding nucleic acid molecules, e.g., DNA,into vectors, e.g., viral vectors, to mediate genome modification invivo may include:

-   -   To achieve NHEJ-mediated gene knockout:    -   Single virus vector:    -   Vector containing two or more expression cassettes:    -   Promoter-Cpf1 coding nucleic acid molecule-terminator    -   Promoter-gRNA1-terminator    -   Promoter-gRNA2-terminator    -   Promoter-gRNA(N)-terminator (up to size limit of vector)    -   Double virus vector:    -   Vector 1 containing one expression cassette for driving the        expression of Cpf1    -   Promoter-Cpf1 coding nucleic acid molecule-terminator    -   Vector 2 containing one more expression cassettes for driving        the expression of one or more guideRNAs    -   Promoter-gRNA1-terminator    -   Promoter-gRNA(N)-terminator (up to size limit of vector)    -   To mediate homology-directed repair.    -   In addition to the single and double virus vector approaches        described above, an additional vector can be used to deliver a        homology-direct repair template.

The promoter used to drive Cpf1 coding nucleic acid molecule expressioncan include:

-   -   AAV ITR can serve as a promoter: this is advantageous for        eliminating the need for an additional promoter element (which        can take up space in the vector). The additional space freed up        can be used to drive the expression of additional elements        (gRNA, etc.). Also, ITR activity is relatively weaker, so can be        used to reduce potential toxicity due to over expression of        Cpf1.    -   For ubiquitous expression, promoters that can be used include:        CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc.

For brain or other CNS expression, can use promoters: SynapsinI for allneurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT forGABAergic neurons, etc.

For liver expression, can use Albumin promoter.

For lung expression, can use SP-B.

For endothelial cells, can use ICAM.

For hematopoietic cells can use IFNbeta or CD45.

For Osteoblasts can one can use the OG-2.

The promoter used to drive guide RNA can include:

-   -   Pol III promoters such as U6 or H1    -   Use of Pol II promoter and intronic cassettes to express gRNA

Adeno Associated Virus (AA V)

Cpf1 and one or more guide RNA can be delivered using adeno associatedvirus (AAV), lentivirus, adenovirus or other plasmid or viral vectortypes, in particular, using formulations and doses from, for example,U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat.No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946(formulations, doses for DNA plasmids) and from clinical trials andpublications regarding the clinical trials involving lentivirus, AAV andadenovirus. For examples, for AAV, the route of administration,formulation and dose can be as in U.S. Pat. No. 8,454,972 and as inclinical trials involving AAV. For Adenovirus, the route ofadministration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g. a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specific genomemodification, the expression of Cpf1 can be driven by a cell-typespecific promoter. For example, liver-specific expression might use theAlbumin promoter and neuron-specific expression (e.g. for targeting CNSdisorders) might use the Synapsin I promoter.

In terms of in vivo delivery, AAV is advantageous over other viralvectors for a couple of reasons:

-   -   Low toxicity (this may be due to the purification method not        requiring ultra centrifugation of cell particles that can        activate the immune response) and    -   Low probability of causing insertional mutagenesis because it        doesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that Cpf1 aswell as a promoter and transcription terminator have to be all fit intothe same viral vector. Constructs larger than 4.5 or 4.75 Kb will leadto significantly reduced virus production. SpCas9 is quite large, thegene itself is over 4.1 Kb, which makes it difficult for packing intoAAV. Therefore embodiments of the invention include utilizing homologsof Cpf1 that are shorter. For example:

Species Cas9 Size (nt) Corynebacter diphtheria 3252 Eubacteriumventriosum 3321 Streptococcus pasteurianus 3390 Lactobacillus farciminis3378 Sphaerochaeta globus 3537 Azospirillum B510 3504 Gluconacetobacterdiazotrophicus 3150 Neisseria cinerea 3246 Roseburia intestinalis 3420Parvibaculum lavamentivorans 3111 Staphylococcus aureus 3159Nitratifractor salsuginis DSM 16511 3396 Campylobacter lari CF89-12 3009Campylobacter jejuni 2952 Streptococcus thermophilus LMD-9 3396rAAV vectors are preferably produced in insect cells, e.g., Spodopterafrugiperda Sf9 insect cells, grown in serum-free suspension culture.Serum-free insect cells can be purchased from commercial vendors, e.g.,Sigma Aldrich (EX-CELL 405).

These species are therefore, in general, preferred Cpf1 species.

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereofOne can select the AAV of the AAV with regard to the cells to betargeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsidAAV1, AAV2, AAV5 or any combination thereof for targeting brain orneuronal cells; and one can select AAV4 for targeting cardiac tissue.AAV8 is useful for delivery to the liver. The herein promoters andvectors are preferred individually. A tabulation of certain AAVserotypes as to these cells (see Grimm, D. et al, J. Virol. 82:5887-5911 (2008)) is as follows:

TABLE 1 Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-713 100 2.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3100 2.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 1000.2 1.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4333 50 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 101.0 0.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.50.1 HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 1251429 ND ND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100ND ND 333 3333 ND ND

Lentivirus

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types.

Lentiviruses may be prepared as follows. After cloning pCasES10 (whichcontains a lentiviral transfer plasmid backbone), HEK293FT at lowpassage (p=5) were seeded in a T-75 flask to 50% confluence the daybefore transfection in DMEM with 10% fetal bovine serum and withoutantibiotics. After 20 hours, media was changed to OptiMEM (serum-free)media and transfection was done 4 hours later. Cells were transfectedwith 10 pg of lentiviral transfer plasmid (pCasES10) and the followingpackaging plasmids: 5 pg of pMD2.G (VSV-g pseudotype), and 7.5 ug ofpsPAX2 (gag/pol/rev/tat). Transfection was done in 4 mL OptiMEM with acationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul Plusreagent). After 6 hours, the media was changed to antibiotic-free DMEMwith 10% fetal bovine serum. These methods use serum during cellculture, but serum-free methods are preferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 um low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 ul of DMEM overnight at 4 C. They were then aliquottedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinfectious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the CRISPR-Cas system of the presentinvention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used/and or adapted to the CRISPR-Cas system of the presentinvention. A minimum of 2.5×106 CD34+ cells per kilogram patient weightmay be collected and prestimulated for 16 to 20 hours in X-VIVO 15medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×106 cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm2 tissue culture flasks coated with fibronectin (25mg/cm2) (RetroNectin, Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571; US20110293571, US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015.

Use of Minimal Promoters

The present application provides a vector for delivering an effectorprotein and at least one CRISPR guide RNA to a cell comprising a minimalpromoter operably linked to a polynucleotide sequence encoding theeffector protein and a second minimal promoter operably linked to apolynucleotide sequence encoding at least one guide RNA, wherein thelength of the vector sequence comprising the minimal promoters andpolynucleotide sequences is less than 4.4Kb. In an embodiment, thevector is an AAV vector. In another embodiment, the effector protein isa CRISPR anzyme. In a further embodiment, the CRISPR enzyme is SaCas9,Cpf1, Cas13b or C2c2.

In a related aspect, the invention provides a lentiviral vector fordelivering an effector protein and at least one CRISPR guide RNA to acell comprising a promoter operably linked to a polynucleotide sequenceencoding Cpf1 and a second promoter operably linked to a polynucleotidesequence encoding at least one guide RNA, wherein the polynucleotidesequences are in reverse orientation.

In another aspect, the invention provides a method of expressing aneffector protein and guide RNA in a cell comprising introducing thevector according any of the vector delivery systems disclosed herein. Inan embodiment of the vector for delivering an effector protein, theminimal promoter is the Mecp2 promoter, tRNA promoter, or U6. In afurther embodiment, the minimal promoter is tissue specific.

Dosage of Vectors

In some embodiments, the vector, e.g., plasmid or viral vector isdelivered to the tissue of interest by, for example, an intramuscularinjection, while other times the delivery is via intravenous,transdermal, intranasal, oral, mucosal, or other delivery methods. Suchdelivery may be either via a single dose, or multiple doses. One skilledin the art understands that the actual dosage to be delivered herein mayvary greatly depending upon a variety of factors, such as the vectorchoice, the target cell, organism, or tissue, the general condition ofthe subject to be treated, the degree of transformation/modificationsought, the administration route, the administration mode, the type oftransformation/modification sought, etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×105 particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×106 particles(for example, about 1×106-1×1012 particles), more preferably at leastabout 1×107 particles, more preferably at least about 1×108 particles(e.g., about 1×108-1×1011 particles or about 1×108-1×1012 particles),and most preferably at least about 1×100 particles (e.g., about 1×109-1x 1010 particles or about 1×109-1×1012 particles), or even at leastabout 1×1010 particles (e.g., about 1×1010-1×1012 particles) of theadenoviral vector. Alternatively, the dose comprises no more than about1×1014 particles, preferably no more than about 1×1013 particles, evenmore preferably no more than about 1×1012 particles, even morepreferably no more than about 1 x 1011 particles, and most preferably nomore than about 1×1010 particles (e.g., no more than about 1×109articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×106 particle units (pu), about 2×106 pu,about 4×106 pu, about 1×107 pu, about 2 x 107 pu, about 4×107 pu, about1×108 pu, about 2×108 pu, about 4×108 pu, about 1×109 pu, about 2×109pu, about 4×109 pu, about 1×1010 pu, about 2×1010 pu, about 4×1010 pu,about 1×1011 pu, about 2×1011 pu, about 4×1011 pu, about 1×1012 pu,about 2×1012 pu, or about 4×1012 pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×1010 to about 1×1010 functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×105 to 1×1050 genomes AAV,from about 1×108 to 1×1020 genomes AAV, from about 1×1010 to about1×1016 genomes, or about 1×1011 to about 1×1016 genomes AAV. A humandosage may be about 1×1013 genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding a CRISPRenzyme, operably linked to said promoter; (iii) a selectable marker;(iv) an origin of replication; and (v) a transcription terminatordownstream of and operably linked to (ii). The plasmid can also encodethe RNA components of a CRISPR complex, but one or more of these mayinstead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

The dosage used for the compositions provided herein include dosages forrepeated administration or repeat dosing. In particular embodiments, theadministration is repeated within a period of several weeks, months, oryears. Suitable assays can be performed to obtain an optimal dosageregime. Repeated administration can allow the use of lower dosage, whichcan positively affect off-target modifications.

RNA Delivery

In particular embodiments, RNA based delivery is used. In theseembodiments, mRNA of the CRISPR effector protein is delivered togetherwith in vitro transcribed guide RNA. Liang et al. describes efficientgenome editing using RNA based delivery (Protein Cell. 2015 May; 6(5):363-372).

RNA delivery: The CRISPR enzyme, for instance a Cpf1, and/or any of thepresent RNAs, for instance a guide RNA, can also be delivered in theform of RNA. Cpf1 mRNA can be generated using in vitro transcription.For example, Cpf1 mRNA can be synthesized using a PCR cassettecontaining the following elements: T7_promoter-kozak sequence(GCCACC)-Cpf1-3′ UTR from beta globin-polyA tail (a string of 120 ormore adenines). The cassette can be used for transcription by T7polymerase. Guide RNAs can also be transcribed using in vitrotranscription from a cassette containing T7_promoter-GG-guide RNAsequence.

To enhance expression and reduce possible toxicity, the CRISPRenzyme-coding sequence and/or the guide RNA can be modified to includeone or more modified nucleoside e.g. using pseudo-U or 5-Methyl-C.

mRNA delivery methods are especially promising for liver deliverycurrently.

Much clinical work on RNA delivery has focused on RNAi or antisense, butthese systems can be adapted for delivery of RNA for implementing thepresent invention. References below to RNAi etc. should be readaccordingly.

CRISPR enzyme mRNA and guide RNA might also be delivered separately.CRISPR enzyme mRNA can be delivered prior to the guide RNA to give timefor CRISPR enzyme to be expressed. CRISPR enzyme mRNA might beadministered 1-12 hours (preferably around 2-6 hours) prior to theadministration of guide RNA.

Alternatively, CRISPR enzyme mRNA and guide RNA can be administeredtogether. Advantageously, a second booster dose of guide RNA can beadministered 1-12 hours (preferably around 2-6 hours) after the initialadministration of CRISPR enzyme mRNA+guide RNA.

RNP

In particular embodiments, pre-complexed guide RNA and CRISPR effectorprotein are delivered as a ribonucleoprotein (RNP). RNPs have theadvantage that they lead to rapid editing effects even more so than theRNA method because this process avoids the need for transcription. Animportant advantage is that both RNP delivery is transient, reducingoff-target effects and toxicity issues. Efficient genome editing indifferent cell types has been observed by Kim et al. (2014, Genome Res.24(6):1012-9), Paix et al. (2015, Genetics 204(1):47-54), Chu et al.(2016, BMC Biotechnol. 16:4), and Wang et al. (2013, Cell. 9;153(4):910-8).

In particular embodiments, the ribonucleoprotein is delivered by way ofa polypeptide-based shuttle agent as described in WO2016161516.WO2016161516 describes efficient transduction of polypeptide cargosusing synthetic peptides comprising an endosome leakage domain (ELD)operably linked to a cell penetrating domain (CPD), to a histidine-richdomain and a CPD. Similarly these polypeptides can be used for thedelivery of CRISPR-effector based RNPs in eukaryotic cells.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver Cpf1 and gRNA (and, for instance, HR repairtemplate) into cells using liposomes or particles. Thus delivery of theCRISPR enzyme, such as a Cpf1 and/or delivery of the RNAs of theinvention may be in RNA form and via microvesicles, liposomes orparticles. For example, Cpf1 mRNA and gRNA can be packaged intoliposomal particles for delivery in vivo. Liposomal transfectionreagents such as lipofectamine from Life Technologies and other reagentson the market can effectively deliver RNA molecules into the liver.

Means of delivery of RNA also preferred include delivery of RNA viananoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei,Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticlesfor small interfering RNA delivery to endothelial cells, AdvancedFunctional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A.,Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-basednanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267:9-21, 2010, PMID: 20059641). Indeed, exosomes have been shown to beparticularly useful in delivery siRNA, a system with some parallels tothe CRISPR system. For instance, El-Andaloussi S, et al.(“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc.2012 December; 7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012Nov. 15.) describe how exosomes are promising tools for drug deliveryacross different biological barriers and can be harnessed for deliveryof siRNA in vitro and in vivo. Their approach is to generate targetedexosomes through transfection of an expression vector, comprising anexosomal protein fused with a peptide ligand. The exosomes are thenpurified and characterized from transfected cell supernatant, then RNAis loaded into the exosomes. Delivery or administration according to theinvention can be performed with exosomes, in particular but not limitedto the brain. Vitamin E (a-tocopherol) may be conjugated with CRISPR Casand delivered to the brain along with high density lipoprotein (HDL),for example in a similar manner as was done by Uno et al. (HUMAN GENETHERAPY 22:711-719 (June 2011)) for delivering short-interfering RNA(siRNA) to the brain. Mice were infused via Osmotic minipumps (model1007D; Alzet, Cupertino, Calif.) filled with phosphate-buffered saline(PBS) or free TocsiBACE or Toc-siBACE/HDL and connected with BrainInfusion Kit 3 (Alzet). A brain-infusion cannula was placed about 0.5 mmposterior to the bregma at midline for infusion into the dorsal thirdventricle. Uno et al. found that as little as 3 nmol of Toc-siRNA withHDL could induce a target reduction in comparable degree by the same ICVinfusion method. A similar dosage of CRISPR Cas conjugated toa-tocopherol and co-administered with HDL targeted to the brain may becontemplated for humans in the present invention, for example, about 3nmol to about 3 μmol of CRISPR Cas targeted to the brain may becontemplated.

Zou et al. ((HUMAN GENE THERAPY 22:465-475 (April 2011)) describes amethod of lentiviral-mediated delivery of short-hairpin RNAs targetingPKCy for in vivo gene silencing in the spinal cord of rats. Zou et al.administered about 10 μl of a recombinant lentivirus having a titer of1×109 transducing units (TU)/ml by an intrathecal catheter. A similardosage of CRISPR Cas expressed in a lentiviral vector may becontemplated for humans in the present invention, for example, about10-50 ml of CRISPR Cas in a lentivirus having a titer of 1×109transducing units (TU)/ml may be contemplated. A similar dosage ofCRISPR Cas expressed in a lentiviral vector targeted to the brain may becontemplated for humans in the present invention, for example, about10-50 ml of CRISPR Cas targeted to the brain in a lentivirus having atiter of 1×109 transducing units (TU)/ml may be contemplated.

Anderson et al. (US 20170079916) provides a modified dendrimernanoparticle for the delivery of therapeutic, prophylactic and/ordiagnostic agents to a subject, comprising: one or more zero to sevengeneration alkylated dendrimers; one or more amphiphilic polymers; andone or more therapeutic, prophylactic and/or diagnostic agentsencapsulated therein. One alkylated dendrimer may be selected from thegroup consisting of poly(ethyleneimine), poly(polyproylenimine),diaminobutane amine polypropylenimine tetramine and poly(amido amine).The therapeutic, prophylactic and diagnostic agent may be selected fromthe group consisting of proteins, peptides, carbohydrates, nucleicacids, lipids, small molecules and combinations thereof.

Anderson et al. (US 20160367686) provides a compound of Formula (I):

and salts thereof, wherein each instance of R L is independentlyoptionally substituted C6-C40 alkenyl, and a composition for thedelivery of an agent to a subject or cell comprising the compound, or asalt thereof, an agent; and optionally, an excipient. The agent may bean organic molecule, inorganic molecule, nucleic acid, protein, peptide,polynucleotide, targeting agent, an isotopically labeled chemicalcompound, vaccine, an immunological agent, or an agent useful inbioprocessing. The composition may further comprise cholesterol, aPEGylated lipid, a phospholipid, or an apolipoprotein.

Anderson et al. (US20150232883) provides a delivery particleformulations and/or systems, preferably nanoparticle deliveryformulations and/or systems, comprising (a) a CRISPR-Cas system RNApolynucleotide sequence; or (b) Cas9; or (c) both a CRISPR-Cas systemRNA polynucleotide sequence and Cas9; or (d) one or more vectors thatcontain nucleic acid molecule(s) encoding (a), (b) or (c), wherein theCRISPR-Cas system RNA polynucleotide sequence and the Cas9 do notnaturally occur together. The delivery particle formulations may furthercomprise a surfactant, lipid or protein, wherein the surfactant maycomprise a cationic lipid.

Anderson et al. (US20050123596) provides examples of microparticles thatare designed to release their payload when exposed to acidic conditions,wherein the microparticles comprise at least one agent to be delivered,a pH triggering agent, and a polymer, wherein the polymer is selectedfrom the group of polymethacrylates and polyacrylates.

Anderson et al (US 20020150626) provides lipid-protein-sugar particlesfor delivery of nucleic acids, wherein the polynucleotide isencapsulated in a lipid-protein-sugar matrix by contacting thepolynucleotide with a lipid, a protein, and a sugar; and spray dryingmixture of the polynucleotide, the lipid, the protein, and the sugar tomake microparticles.

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g. byinjection. Injection can be performed stereotactically via a craniotomy.

Enhancing NHEJ or HR efficiency is also helpful for delivery. It ispreferred that NHEJ efficiency is enhanced by co-expressingend-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011August; 188(4): 787-797). It is preferred that HR efficiency isincreased by transiently inhibiting NHEJ machineries such as Ku70 andKu86. HR efficiency can also be increased by co-expressing prokaryoticor eukaryotic homologous recombination enzymes such as RecBCD, RecA.

Particles

In some aspects or embodiments, a composition comprising a deliveryparticle formulation may be used. In some aspects or embodiments, theformulation comprises a CRISPR complex, the complex comprising a CRISPRprotein and—a guide which directs sequence-specific binding of theCRISPR complex to a target sequence. In some embodiments, the deliveryparticle comprises a lipid-based particle, optionally a lipidnanoparticle, or cationic lipid and optionally biodegradable polymer. Insome embodiments, the cationic lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In some embodiments,the hydrophilic polymer comprises ethylene glycol or polyethyleneglycol. In some embodiments, the delivery particle further comprises alipoprotein, preferably cholesterol. In some embodiments, the deliveryparticles are less than 500 nm in diameter, optionally less than 250 nmin diameter, optionally less than 100 nm in diameter, optionally about35 nm to about 60 nm in diameter.

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter. Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.diameter) of less than 100 microns (pm). In some embodiments, inventiveparticles have a greatest dimension of less than 10 p m. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

In terms of this invention, it is preferred to have one or morecomponents of CRISPR complex, e.g., CRISPR enzyme or mRNA or guide RNAdelivered using nanoparticles or lipid envelopes. Other delivery systemsor vectors are may be used in conjunction with the nanoparticle aspectsof the invention.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In certain preferred embodiments, nanoparticles ofthe invention have a greatest dimension (e.g., diameter) of 500 nm orless. In other preferred embodiments, nanoparticles of the inventionhave a greatest dimension ranging between 25 nm and 200 nm. In otherpreferred embodiments, nanoparticles of the invention have a greatestdimension of 100 nm or less. In other preferred embodiments,nanoparticles of the invention have a greatest dimension ranging between35 nm and 60 nm. It will be appreciated that reference made herein toparticles or nanoparticles can be interchangeable, where appropriate.

It will be understood that the size of the particle will differdepending as to whether it is measured before or after loading.Accordingly, in particular embodiments, the term “nanoparticles” mayapply only to the particles pre loading.

Nanoparticles encompassed in the present invention may be provided indifferent forms, e.g., as solid nanoparticles (e.g., metal such assilver, gold, iron, titanium), non-metal, lipid-based solids, polymers),suspensions of nanoparticles, or combinations thereof. Metal,dielectric, and semiconductor nanoparticles may be prepared, as well ashybrid structures (e.g., core-shell nanoparticles). Nanoparticles madeof semiconducting material may also be labeled quantum dots if they aresmall enough (typically sub 10 nm) that quantization of electronicenergy levels occurs. Such nanoscale particles are used in biomedicalapplications as drug carriers or imaging agents and may be adapted forsimilar purposes in the present invention.

Semi-solid and soft nanoparticles have been manufactured, and are withinthe scope of the present invention. A prototype nanoparticle ofsemi-solid nature is the liposome. Various types of liposomenanoparticles are currently used clinically as delivery systems foranticancer drugs and vaccines. Nanoparticles with one half hydrophilicand the other half hydrophobic are termed Janus particles and areparticularly effective for stabilizing emulsions. They can self-assembleat water/oil interfaces and act as solid surfactants.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry(MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarization interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of CRISPR-Cassystem e.g., CRISPR enzyme or mRNA or guide RNA, or any combinationthereof, and may include additional carriers and/or excipients) toprovide particles of an optimal size for delivery for any in vitro, exvivo and/or in vivo application of the present invention. In certainpreferred embodiments, particle dimension (e.g., diameter)characterization is based on measurements using dynamic laser scattering(DLS). Mention is made of U.S. Pat. Nos. 8,709,843; 6,007,845;5,855,913; 5,985,309; 5,543,158; and the publication by James E. Dahlmanand Carmen Barnes et al. Nature Nanotechnology (2014) published online11 May 2014, doi:10.1038/nnano.2014.84, concerning particles, methods ofmaking and using them and measurements thereof.

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to solid, semi-solid,emulsion, or colloidal particles. As such any of the delivery systemsdescribed herein, including but not limited to, e.g., lipid-basedsystems, liposomes, micelles, microvesicles, exosomes, or gene gun maybe provided as particle delivery systems within the scope of the presentinvention.

CRISPR enzyme mRNA and guide RNA may be delivered simultaneously usingparticles or lipid envelopes; for instance, CRISPR enzyme and RNA of theinvention, e.g., as a complex, can be delivered via a particle as inDahlman et al., WO2015089419 A2 and documents cited therein, such as 7C1(see, e.g., James E. Dahlman and Carmen Barnes et al. NatureNanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84), e.g., delivery particle comprising lipid orlipidoid and hydrophilic polymer, e.g., cationic lipid and hydrophilicpolymer, for instance wherein the cationic lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or whereinthe hydrophilic polymer comprises ethylene glycol or polyethylene glycol(PEG); and/or wherein the particle further comprises cholesterol (e.g.,particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0;formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0;formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5), whereinparticles are formed using an efficient, multistep process whereinfirst, effector protein and RNA are mixed together, e.g., at a 1:1 molarratio, e.g., at room temperature, e.g., for 30 minutes, e.g., insterile, nuclease free 1×PBS; and separately, DOTAP, DMPC, PEG, andcholesterol as applicable for the formulation are dissolved in alcohol,e.g., 100% ethanol; and, the two solutions are mixed together to formparticles containing the complexes).

Nucleic acid-targeting effector proteins (such as a Type V protein suchCpf1) mRNA and guide RNA may be delivered simultaneously using particlesor lipid envelopes. Examples of suitable particles include but are notlimited to those described in U.S. Pat. No. 9,301,923.

For example, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and invivo mRNA delivery using lipid-enveloped pH-responsive polymernanoparticles” Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable core-shellstructured nanoparticles with a poly(β-amino ester) (PBAE) coreenveloped by a phospholipid bilayer shell. These were developed for invivo mRNA delivery. The pH-responsive PBAE component was chosen topromote endosome disruption, while the lipid surface layer was selectedto minimize toxicity of the polycation core. Such are, therefore,preferred for delivering RNA of the present invention.

Liu et al. (US 20110212179) provides bimodal porous polymer microspherescomprising a base polymer, wherein the particle comprises macroporeshaving a diameter ranging from about 20 to about 500 microns andmicropores having a diameter ranging from about 1 to about 70 microns,and wherein the microspheres have a diameter ranging from about 50 toabout 1100 microns.

Berg et al. (US20160174546) a nanolipid delivery system, in particular anano-particle concentrate, comprising: a composition comprising a lipid,oil or solvent, the composition having a viscosity of less than 100 cPat 25.degree. C. and a Kauri Butanol solvency of greater than 25 Kb; andat least one amphipathic compound selected from the group consisting ofan alkoxylated lipid, an alkoxylated fatty acid, an alkoxylated alcohol,a heteroatomic hydrophilic lipid, a heteroatomic hydrophilic fatty acid,a heteroatomic hydrophilic alcohol, a diluent, and combinations thereof,wherein the compound is derived from a starting compound having aviscosity of less than 1000 cP at 50.degree. C., wherein the concentrateis configured to provide a stable nano emulsion having a D50 and a meanaverage particle size distribution of less than 100 nm when diluted.

Liu et al. (US 20140301951) provides a protocell nanostructurecomprising: a porous particle core comprising a plurality of pores; andat least one lipid bilayer surrounding the porous particle core to forma protocell, wherein the protocell is capable of loading one or morecargo components to the plurality of pores of the porous particle coreand releasing the one or more cargo components from the porous particlecore across the surrounding lipid bilayer.

Chromy et al. (US 20150105538) provides methods and systems forassembling, solubilizing and/or purifying a membrane associated proteinin a nanolipoprotein particle, which comprise a temperature transitioncycle performed in presence of a detergent, wherein during thetemperature transition cycle the nanolipoprotein components are broughtto a temperature above and below the gel to liquid crystallingtransition temperature of the membrane forming lipid of thenanolipoprotein particle.

Bader et al. (US 20150250725), provides a method for producing a lipidparticle comprising the following: i) providing a first solutioncomprising denatured apolipoprotein, ii) adding the first solution to asecond solution comprising at least two lipids and a detergent but noapolipoprotein, and iii) removing the detergent from the solutionobtained in ii) and thereby producing a lipid particle.

Mirkin et al., (US20100129793) provides a method of preparing acomposite particle comprising the steps of (a) admixing a dielectriccomponent and a magnetic component to form a first intermediate, (b)admixing the first intermediate and gold seeds to form a secondintermediate, and (c) forming a gold shell on the second intermediate byadmixing the second intermediate with a gold source and a reducing agentto form said composite particle.

In one embodiment, particles/nanoparticles based on self assemblingbioadhesive polymers are contemplated, which may be applied to oraldelivery of peptides, intravenous delivery of peptides and nasaldelivery of peptides, all to the brain. Other embodiments, such as oralabsorption and ocular delivery of hydrophobic drugs are alsocontemplated. The molecular envelope technology involves an engineeredpolymer envelope which is protected and delivered to the site of thedisease (see, e.g., Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026;Siew, A., et al. Mol Pharm, 2012. 9(1):14-28; Lalatsa, A., et al. JContr Rel, 2012. 161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012.9(6):1665-80; Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74;Garrett, N. L., et al. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N.L., et al. J Raman Spect, 2012. 43(5):681-688; Ahmad, S., et al. J RoyalSoc Interface 2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv,2006. 3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9and Uchegbu, I. F., et al. Int J Pharm, 2001. 224:185-199). Doses ofabout 5 mg/kg are contemplated, with single or multiple doses, dependingon the target tissue.

In one embodiment, particles/nanoparticles that can deliver RNA to acancer cell to stop tumor growth developed by Dan Anderson's lab at MITmay be used/and or adapted to the CRISPR Cas system of the presentinvention. In particular, the Anderson lab developed fully automated,combinatorial systems for the synthesis, purification, characterization,and formulation of new biomaterials and nanoformulations. See, e.g.,Alabi et al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110(32):12881-6;Zhang et al., Adv Mater. 2013 Sep. 6; 25(33):4641-5; Jiang et al., NanoLett. 2013 Mar. 13; 13(3):1059-64; Karagiannis et al., ACS Nano. 2012Oct. 23; 6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28;6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93.

The lipid particles developed by the Qiaobing Xu's lab at TuftsUniversity may be used/adapted to the present delivery system for cancertherapy. See Wang et al., J. Control Release, 2017 Jan. 31. pii:S0168-3659(17)30038-X. doi: 10.1016/j.jconrel.2017.01.037. [Epub aheadof print]; Altmoglu et al., Biomater Sci., 4(12):1773-80, Nov. 15, 2016;Wang et al., PNAS, 113(11):2868-73 Mar. 15, 2016; Wang et al., PloS One,10(11): e0141860. doi: 10.1371/journal.pone.0141860. eCollection 2015,Nov. 3, 2015; Takeda et al., Neural Regen Res. 10(5):689-90, May 2015;Wang et al., Adv. Healthc Mater., 3(9):1398-403, Sep. 2014; and Wang etal., Agnew Chem Int Ed Engl., 53(11):2893-8, Mar. 10, 2014.

US patent application 20110293703 relates to lipidoid compounds are alsoparticularly useful in the administration of polynucleotides, which maybe applied to deliver the CRISPR Cas system of the present invention. Inone aspect, the aminoalcohol lipidoid compounds are combined with anagent to be delivered to a cell or a subject to form microparticles,nanoparticles, liposomes, or micelles. The agent to be delivered by theparticles, liposomes, or micelles may be in the form of a gas, liquid,or solid, and the agent may be a polynucleotide, protein, peptide, orsmall molecule. The aminoalcohol lipidoid compounds may be combined withother aminoalcohol lipidoid compounds, polymers (synthetic or natural),surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to formthe particles. These particles may then optionally be combined with apharmaceutical excipient to form a pharmaceutical composition.

US Patent Publication No. 20110293703 also provides methods of preparingthe aminoalcohol lipidoid compounds. One or more equivalents of an amineare allowed to react with one or more equivalents of anepoxide-terminated compound under suitable conditions to form anaminoalcohol lipidoid compound of the present invention. In certainembodiments, all the amino groups of the amine are fully reacted withthe epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30-100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell.

US Patent Publication No. 20130302401 relates to a class ofpoly(beta-amino alcohols) (PBAAs) has been prepared using combinatorialpolymerization. The inventive PBAAs may be used in biotechnology andbiomedical applications as coatings (such as coatings of films ormultilayer films for medical devices or implants), additives, materials,excipients, non-biofouling agents, micropatterning agents, and cellularencapsulation agents. When used as surface coatings, these PBAAselicited different levels of inflammation, both in vitro and in vivo,depending on their chemical structures. The large chemical diversity ofthis class of materials allowed us to identify polymer coatings thatinhibit macrophage activation in vitro. Furthermore, these coatingsreduce the recruitment of inflammatory cells, and reduce fibrosis,following the subcutaneous implantation of carboxylated polystyrenemicroparticles. These polymers may be used to form polyelectrolytecomplex capsules for cell encapsulation. The invention may also havemany other biological applications such as antimicrobial coatings, DNAor siRNA delivery, and stem cell tissue engineering. The teachings of USPatent Publication No. 20130302401 may be applied to the CRISPR Cassystem of the present invention.

In another embodiment, lipid nanoparticles (LNPs) are contemplated. Anantitransthyretin small interfering RNA has been encapsulated in lipidnanoparticles and delivered to humans (see, e.g., Coelho et al., N EnglJ Med 2013; 369:819-29), and such a system may be adapted and applied tothe CRISPR Cas system of the present invention. Doses of about 0.01 toabout 1 mg per kg of body weight administered intravenously arecontemplated. Medications to reduce the risk of infusion-relatedreactions are contemplated, such as dexamethasone, acetampinophen,diphenhydramine or cetirizine, and ranitidine are contemplated. Multipledoses of about 0.3 mg per kilogram every 4 weeks for five doses are alsocontemplated.

Zhu et al. (US20140348900) provides for a process for preparingliposomes, lipid discs, and other lipid nanoparticles using a multi-portmanifold, wherein the lipid solution stream, containing an organicsolvent, is mixed with two or more streams of aqueous solution (e.g.,buffer). In some aspects, at least some of the streams of the lipid andaqueous solutions are not directly opposite of each other. Thus, theprocess does not require dilution of the organic solvent as anadditional step. In some embodiments, one of the solutions may alsocontain an active pharmaceutical ingredient (API). This inventionprovides a robust process of liposome manufacturing with different lipidformulations and different payloads. Particle size, morphology, and themanufacturing scale can be controlled by altering the port size andnumber of the manifold ports, and by selecting the flow rate or flowvelocity of the lipid and aqueous solutions.

Cullis et al. (US 20140328759) provides limit size lipid nanoparticleswith a diameter from 10-100 nm, in particular comprising a lipid bilayersurrounding an aqueous core. Methods and apparatus for preparing suchlimit size lipid nanoparticles are also disclosed.

Manoharan et al. (US 20140308304) provides cationic lipids of formula(I)

or a salt thereof, wherein X is N or P; R′ is absent, hydrogen, oralkyl; with respect to R1 and R2, (i) R1 and R2 are each, independently,optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkylalkyl, heterocycle or R10; (ii) R1 and R2, together with thenitrogen atom to which they are attached, form an optionally substitutedheterocyclic ring; or (iii) one of R1 and R2 is optionally substitutedalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle,and the other forms a 4-10 member heterocyclic ring or heteroaryl with(a) the adjacent nitrogen atom and (b) the (R)a group adjacent to thenitrogen atom; each occurrence of R is, independently, —(CR3R4)-; eachoccurrence of R3 and R4 are, independently H, halogen, OH, alkyl,alkoxy, —NH.sub.2, alkylamino, or dialkylamino; or R3 and R4, togetherwith the carbon atom to which they are directly attached, form acycloalkyl group, wherein no more than three R groups in each chainattached to the atom X* are cycloalkyl; each occurrence of R.sup.10 isindependently selected from PEG and polymers based on poly(oxazoline),poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol),poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] andpoly(amino acid)s, wherein (i) the PEG or polymer is linear or branched,(ii) the PEG or polymer is polymerized by n subunits, (iii) n is anumber-averaged degree of polymerization between 10 and 200 units, and(iv) wherein the compound of formula has at most two R10 groups; Q isabsent or is —O—, —NH—, —S—, —C(O)O—, —OC(O)—, —C(O)N(R4)-, —N(R5)C(O)—,—S—S—, —OC(O)O—, —O—N.dbd.C(R5)-, —C(R5).dbd.N—O—, —OC(O)N(R5)-,—N(R5)C(O)N(R5)-, —N(R5)C(O)O—, —C(O)S—, —C(S)O— or—C(R5).dbd.N—O—C(O)—; Q1 and Q2 are each, independently, absent, —O—,—S—, —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—,—C(O)(NR5)-, —N(R5)C(O)—, —C(S)(NR5)-, —N(R5)C(O)—, —N(R5)C(O)N(R5)-, or—OC(O)O—; Q3 and Q4 are each, independently, H, —(CR3R4)-, aryl, or acholesterol moiety; each occurrence of A1, A2, A3 and A4 is,independently, —(CR5R5-CR5.dbd.CR5)-; each occurrence of R5 is,independently, H or alkyl; M1 and M2 are each, independently, abiodegradable group (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—,—C(S)O—, —S—S—, —C(R5).dbd.N—, —N.dbd.C(R5)-, —C(R5).dbd.N—O—,—O—N.dbd.C(R5)-, —C(O)(NR5)-, —N(R5)C(O)—, —C(S)(NR5)-, —N(R5)C(O)—,—N(R5)C(O)N(R5)-, —OC(O)O—, —OSi(R5).sub.2O—, —C(O)(CR3R4)C(O)O—, or—OC(O)(CR3R4)C(O)—); Z is absent, alkylene or —O—P(O)(OH)—O—; each -----attached to Z is an optional bond, such that when Z is absent, Q3 and Q4are not directly covalently bound together; a is 1, 2, 3, 4, 5 or 6; bis 0, 1, 2, or 3; c, d, e, f, i, j, m, n, q and r are each,independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; g and h are each,independently, 0, 1 or 2; k and l are each, independently, 0 or 1, whereat least one of k and l is 1; and o and p are each, independently, 0, 1or 2, wherein Q3 and Q4 are each, independently, separated from thetertiary atom marked with an asterisk (X*) by a chain of 8 or moreatoms. The cationic lipid can be used with other lipid components suchas cholesterol and PEG-lipids to form lipid nanoparticles witholigonucleotides, to facilitate the cellular uptake and endosomalescape, and to knockdown target mRNA both in vitro and in vivo.

LNPs have been shown to be highly effective in delivering siRNAs to theliver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No. 4, pages 363-470) and are therefore contemplated for deliveringRNA encoding CRISPR Cas to the liver. A dosage of about four doses of 6mg/kg of the LNP every two weeks may be contemplated. Tabernero et al.demonstrated that tumor regression was observed after the first 2 cyclesof LNPs dosed at 0.7 mg/kg, and by the end of 6 cycles the patient hadachieved a partial response with complete regression of the lymph nodemetastasis and substantial shrinkage of the liver tumors. A completeresponse was obtained after 40 doses in this patient, who has remainedin remission and completed treatment after receiving doses over 26months. Two patients with RCC and extrahepatic sites of diseaseincluding kidney, lung, and lymph nodes that were progressing followingprior therapy with VEGF pathway inhibitors had stable disease at allsites for approximately 8 to 12 months, and a patient with PNET andliver metastases continued on the extension study for 18 months (36doses) with stable disease.

However, the charge of the LNP must be taken into consideration. Ascationic lipids combined with negatively charged lipids to inducenonbilayer structures that facilitate intracellular delivery. Becausecharged LNPs are rapidly cleared from circulation following intravenousinjection, ionizable cationic lipids with pKa values below 7 weredeveloped (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12,pages 1286-2200, Dec. 2011). Negatively charged polymers such as RNA maybe loaded into LNPs at low pH values (e.g., pH 4) where the ionizablelipids display a positive charge. However, at physiological pH values,the LNPs exhibit a low surface charge compatible with longer circulationtimes. Four species of ionizable cationic lipids have been focused upon,namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).It has been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, Dec. 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR Cas encapsulation may be used/and oradapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, Dec. 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(o-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificCRISPR Cas RNA may be encapsulated in LNPs containing DLinDAP, DLinDMA,DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL:PEGS-DMG orPEG-C-DOMG at 40:10:40:10 molar ratios). When required, 0.2% SP-DiOC18(Invitrogen, Burlington, Canada) may be incorporated to assess cellularuptake, intracellular delivery, and biodistribution. Encapsulation maybe performed by dissolving lipid mixtures comprised of cationiclipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) in ethanolto a final lipid concentration of 10 mmol/l. This ethanol solution oflipid may be added drop-wise to 50 mmol/l citrate, pH 4.0 to formmultilamellar vesicles to produce a final concentration of 30% ethanolvol/vol. Large unilamellar vesicles may be formed following extrusion ofmultilamellar vesicles through two stacked 80 nm Nucleoporepolycarbonate filters using the Extruder (Northern Lipids, Vancouver,Canada). Encapsulation may be achieved by adding RNA dissolved at 2mg/ml in 50 mmol/l citrate, pH 4.0 containing 30% ethanol vol/voldrop-wise to extruded preformed large unilamellar vesicles andincubation at 31° C. for 30 minutes with constant mixing to a finalRNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol andneutralization of formulation buffer were performed by dialysis againstphosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2regenerated cellulose dialysis membranes. Nanoparticle size distributionmay be determined by dynamic light scattering using a NICOMP 370particle sizer, the vesicle/intensity modes, and Gaussian fitting(Nicomp Particle Sizing, Santa Barbara, Calif.). The particle size forall three LNP systems may be ˜70 nm in diameter. RNA encapsulationefficiency may be determined by removal of free RNA using VivaPureDMiniH columns (Sartorius Stedim Biotech) from samples collected beforeand after dialysis. The encapsulated RNA may be extracted from theeluted nanoparticles and quantified at 260 nm. RNA to lipid ratio wasdetermined by measurement of cholesterol content in vesicles using theCholesterol E enzymatic assay from Wako Chemicals USA (Richmond, Va.).In conjunction with the herein discussion of LNPs and PEG lipids,PEGylated liposomes or LNPs are likewise suitable for delivery of aCRISPR-Cas system or components thereof.

Preparation of large LNPs may be used/and or adapted from Rosin et al,Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011. A lipidpremix solution (20.4 mg/ml total lipid concentration) may be preparedin ethanol containing DLinKC2-DMA, DSPC, and cholesterol at 50:10:38.5molar ratios. Sodium acetate may be added to the lipid premix at a molarratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids may besubsequently hydrated by combining the mixture with 1.85 volumes ofcitrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting inspontaneous liposome formation in aqueous buffer containing 35% ethanol.The liposome solution may be incubated at 37° C. to allow fortime-dependent increase in particle size. Aliquots may be removed atvarious times during incubation to investigate changes in liposome sizeby dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments,Worcestershire, UK). Once the desired particle size is achieved, anaqueous PEG lipid solution (stock=10 mg/ml PEG-DMG in 35% (vol/vol)ethanol) may be added to the liposome mixture to yield a final PEG molarconcentration of 3.5% of total lipid. Upon addition of PEG-lipids, theliposomes should their size, effectively quenching further growth. RNAmay then be added to the empty liposomes at an RNA to total lipid ratioof approximately 1:10 (wt:wt), followed by incubation for 30 minutes at37° C. to form loaded LNPs. The mixture may be subsequently dialyzedovernight in PBS and filtered with a 0.45-μm syringe filter.

Preassembled recombinant CRISPR-Cpf1 complexes comprising Cpf1 and crRNAmay be transfected, for example by electroporation, resulting in highmutation rates and absence of detectable off-target mutations. Hur, J.K. et al, Targeted mutagenesis in mice by electroporation of Cpf1ribonucleoproteins, Nat Biotechnol. 2016 Jun. 6. doi: 10.1038/nbt.3596.[Epub ahead of print]

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g. byinjection. Injection can be performed stereotactically via a craniotomy.

Enhancing NHEJ or HR efficiency is also helpful for delivery. It ispreferred that NHEJ efficiency is enhanced by co-expressingend-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011August; 188(4): 787-797). It is preferred that HR efficiency isincreased by transiently inhibiting NHEJ machineries such as Ku70 andKu86. HR efficiency can also be increased by co-expressing prokaryoticor eukaryotic homologous recombination enzymes such as RecBCD, RecA.

In some embodiments, sugar-based particles may be used, for exampleGalNAc, as described herein and with reference to WO2014118272(incorporated herein by reference) and Nair, J K et al., 2014, Journalof the American Chemical Society 136 (49), 16958-16961) and the teachingherein, especially in respect of delivery applies to all particlesunless otherwise apparent. This may be considered to be a sugar-basedparticle and further details on other particle delivery systems and/orformulations are provided herein. GalNAc can therefore be considered tobe a particle in the sense of the other particles described herein, suchthat general uses and other considerations, for instance delivery ofsaid particles, apply to GalNAc particles as well. A solution-phaseconjugation strategy may for example be used to attach triantennaryGalNAc clusters (mol. wt. ˜2000) activated as PFP (pentafluorophenyl)esters onto 5′-hexylamino modified oligonucleotides (5′-HA ASOs, mol.wt. ˜8000 Da; Ostergaard et al., Bioconjugate Chem., 2015, 26 (8), pp1451-1455). Similarly, poly(acrylate) polymers have been described forin vivo nucleic acid delivery (see WO2013158141 incorporated herein byreference). In further alternative embodiments, pre-mixing CRISPRnanoparticles (or protein complexes) with naturally occurring serumproteins may be used in order to improve delivery (Akinc A et al, 2010,Molecular Therapy vol. 18 no. 7, 1357-1364).

Nanoclews

Further, the programmable nucleic acid modifying agents may be deliveredusing nanoclews, for example as described in Sun W et al, Cocoon-likeself-degradable DNA nanoclew for anticancer drug delivery., J Am ChemSoc. 2014 Oct. 22; 136(42):14722-5. doi: 10.1021/ja5088024. Epub 2014Oct. 13. ; or in Sun W et al, Self-Assembled DNA Nanoclews for theEfficient Delivery of CRISPR-Cas9 for Genome Editing., Angew Chem Int EdEngl. 2015 Oct. 5; 54(41):12029-33. doi: 10.1002/anie.201506030. Epub2015 Aug. 27.

LNP

In some embodiments, delivery is by encapsulation of the programmablenucleic acid modifying agents in a lipid particle such as an LNP. Insome embodiments, therefore, lipid nanoparticles (LNPs) arecontemplated. An antitransthyretin small interfering RNA has beenencapsulated in lipid nanoparticles and delivered to humans (see, e.g.,Coelho et al., N Engl J Med 2013; 369:819-29), and such a system may beadapted and applied to the CRISPR Cas system of the present invention.Doses of about 0.01 to about 1 mg per kg of body weight administeredintravenously are contemplated. Medications to reduce the risk ofinfusion-related reactions are contemplated, such as dexamethasone,acetampinophen, diphenhydramine or cetirizine, and ranitidine arecontemplated. Multiple doses of about 0.3 mg per kilogram every 4 weeksfor five doses are also contemplated.

LNPs have been shown to be highly effective in delivering siRNAs to theliver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No. 4, pages 363-470) and are therefore contemplated for deliveringRNA encoding CRISPR Cas to the liver. A dosage of about four doses of 6mg/kg of the LNP every two weeks may be contemplated. Tabernero et al.demonstrated that tumor regression was observed after the first 2 cyclesof LNPs dosed at 0.7 mg/kg, and by the end of 6 cycles the patient hadachieved a partial response with complete regression of the lymph nodemetastasis and substantial shrinkage of the liver tumors. A completeresponse was obtained after 40 doses in this patient, who has remainedin remission and completed treatment after receiving doses over 26months. Two patients with RCC and extrahepatic sites of diseaseincluding kidney, lung, and lymph nodes that were progressing followingprior therapy with VEGF pathway inhibitors had stable disease at allsites for approximately 8 to 12 months, and a patient with PNET andliver metastases continued on the extension study for 18 months (36doses) with stable disease.

However, the charge of the LNP must be taken into consideration. Ascationic lipids combined with negatively charged lipids to inducenonbilayer structures that facilitate intracellular delivery. Becausecharged LNPs are rapidly cleared from circulation following intravenousinjection, ionizable cationic lipids with pKa values below 7 weredeveloped (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12,pages 1286-2200, Dec. 2011). Negatively charged polymers such as RNA maybe loaded into LNPs at low pH values (e.g., pH 4) where the ionizablelipids display a positive charge. However, at physiological pH values,the LNPs exhibit a low surface charge compatible with longer circulationtimes. Four species of ionizable cationic lipids have been focused upon,namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).It has been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, Dec. 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR Cas encapsulation may be used/and oradapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, Dec. 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2”-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(o-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificCRISPR Cas RNA may be encapsulated in LNPs containing DLinDAP, DLinDMA,DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL:PEGS-DMG orPEG-C-DOMG at 40:10:40:10 molar ratios). When required, 0.2% SP-DiOC18(Invitrogen, Burlington, Canada) may be incorporated to assess cellularuptake, intracellular delivery, and biodistribution. Encapsulation maybe performed by dissolving lipid mixtures comprised of cationiclipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) in ethanolto a final lipid concentration of 10 mmol/l. This ethanol solution oflipid may be added drop-wise to 50 mmol/l citrate, pH 4.0 to formmultilamellar vesicles to produce a final concentration of 30% ethanolvol/vol. Large unilamellar vesicles may be formed following extrusion ofmultilamellar vesicles through two stacked 80 nm Nuclepore polycarbonatefilters using the Extruder (Northern Lipids, Vancouver, Canada).Encapsulation may be achieved by adding RNA dissolved at 2 mg/ml in 50mmol/l citrate, pH 4.0 containing 30% ethanol vol/vol drop-wise toextruded preformed large unilamellar vesicles and incubation at 31° C.for 30 minutes with constant mixing to a final RNA/lipid weight ratio of0.06/1 wt/wt. Removal of ethanol and neutralization of formulationbuffer were performed by dialysis against phosphate-buffered saline(PBS), pH 7.4 for 16 hours using Spectra/Por 2 regenerated cellulosedialysis membranes. Nanoparticle size distribution may be determined bydynamic light scattering using a NICOMP 370 particle sizer, thevesicle/intensity modes, and Gaussian fitting (Nicomp Particle Sizing,Santa Barbara, Calif.). The particle size for all three LNP systems maybe ˜70 nm in diameter. RNA encapsulation efficiency may be determined byremoval of free RNA using VivaPureD MiniH columns (Sartorius StedimBiotech) from samples collected before and after dialysis. Theencapsulated RNA may be extracted from the eluted nanoparticles andquantified at 260 nm. RNA to lipid ratio was determined by measurementof cholesterol content in vesicles using the Cholesterol E enzymaticassay from Wako Chemicals USA (Richmond, Va.). In conjunction with theherein discussion of LNPs and PEG lipids, PEGylated liposomes or LNPsare likewise suitable for delivery of a CRISPR-Cas system or componentsthereof.

A lipid premix solution (20.4 mg/ml total lipid concentration) may beprepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premixat a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids maybe subsequently hydrated by combining the mixture with 1.85 volumes ofcitrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting inspontaneous liposome formation in aqueous buffer containing 35% ethanol.The liposome solution may be incubated at 37° C. to allow fortime-dependent increase in particle size. Aliquots may be removed atvarious times during incubation to investigate changes in liposome sizeby dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments,Worcestershire, UK). Once the desired particle size is achieved, anaqueous PEG lipid solution (stock=10 mg/ml PEG-DMG in 35% (vol/vol)ethanol) may be added to the liposome mixture to yield a final PEG molarconcentration of 3.5% of total lipid. Upon addition of PEG-lipids, theliposomes should their size, effectively quenching further growth. RNAmay then be added to the empty liposomes at an RNA to total lipid ratioof approximately 1:10 (wt:wt), followed by incubation for 30 minutes at37° C. to form loaded LNPs. The mixture may be subsequently dialyzedovernight in PBS and filtered with a 0.45-μm syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other nanoparticles(particularly gold nanoparticles) are also contemplated as a means todelivery CRISPR-Cas system to intended targets. Significant data showthat AuraSense Therapeutics' Spherical Nucleic Acid (SNA™) constructs,based upon nucleic acid-functionalized gold nanoparticles, are useful.

Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen etal., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling nanoparticles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGF R2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of CRISPR Cas is envisioned for deliveryin the self-assembling nanoparticles of Schiffelers et al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007,vol. 104, no. 39)may also be applied to the present invention. The nanoplexes of Bartlettet al. are prepared by mixing equal volumes of aqueous solutions ofcationic polymer and nucleic acid to give a net molar excess ofionizable nitrogen (polymer) to phosphate (nucleic acid) over the rangeof 2 to 6. The electrostatic interactions between cationic polymers andnucleic acid resulted in the formation of polyplexes with averageparticle size distribution of about 100 nm, hence referred to here asnanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered fromMacrocyclics (Dallas, Tex.). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNAsense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove tracemetal contaminants. Tf-targeted and nontargeted siRNA nanoparticles maybe formed by using cyclodextrin-containing polycations. Typically,nanoparticles were formed in water at a charge ratio of 3 (+/−) and ansiRNA concentration of 0.5 g/liter. One percent of the adamantane-PEGmolecules on the surface of the targeted nanoparticles were modifiedwith Tf (adamantane-PEG-Tf). The nanoparticles were suspended in a 5%(wt/vol) glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA clinicaltrial that uses a targeted nanoparticle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetednanoparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The nanoparticles consist of a synthetic deliverysystem containing: (1) a linear, cyclodextrin-based polymer (CDP), (2) ahuman transferrin protein (TF) targeting ligand displayed on theexterior of the nanoparticle to engage TF receptors (TFR) on the surfaceof the cancer cells, (3) a hydrophilic polymer (polyethylene glycol(PEG) used to promote nanoparticle stability in biological fluids), and(4) siRNA designed to reduce the expression of the RRM2 (sequence usedin the clinic was previously denoted siR2B+5). The TFR has long beenknown to be upregulated in malignant cells, and RRM2 is an establishedanti-cancer target. These nanoparticles (clinical version denoted asCALAA-01) have been shown to be well tolerated in multi-dosing studiesin non-human primates. Although a single patient with chronic myeloidleukaemia has been administered siRNA by liposomal delivery, Davis etal.'s clinical trial is the initial human trial to systemically deliversiRNA with a targeted delivery system and to treat patients with solidcancer. To ascertain whether the targeted delivery system can provideeffective delivery of functional siRNA to human tumors, Davis et al.investigated biopsies from three patients from three different dosingcohorts; patients A, B and C, all of whom had metastatic melanoma andreceived CALAA-01 doses of 18, 24 and 30 mg m-2 siRNA, respectively.Similar doses may also be contemplated for the CRISPR Cas system of thepresent invention. The delivery of the invention may be achieved withnanoparticles containing a linear, cyclodextrin-based polymer (CDP), ahuman transferrin protein (TF) targeting ligand displayed on theexterior of the nanoparticle to engage TF receptors (TFR) on the surfaceof the cancer cells and/or a hydrophilic polymer (for example,polyethylene glycol (PEG) used to promote nanoparticle stability inbiological fluids).

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingpolymer conjugated to a surfactant, hydrophilic polymer or lipid.

U.S. Pat. No. 6,007,845, incorporated herein by reference, providesparticles which have a core of a multiblock copolymer formed bycovalently linking a multifunctional compound with one or morehydrophobic polymers and one or more hydrophilic polymers, and contain abiologically active material.

U.S. Pat. No. 5,855,913, incorporated herein by reference, provides aparticulate composition having aerodynamically light particles having atap density of less than 0.4 g/cm3 with a mean diameter of between 5 μmand 30 μm, incorporating a surfactant on the surface thereof for drugdelivery to the pulmonary system.

U.S. Pat. No. 5,985,309, incorporated herein by reference, providesparticles incorporating a surfactant and/or a hydrophilic or hydrophobiccomplex of a positively or negatively charged therapeutic or diagnosticagent and a charged molecule of opposite charge for delivery to thepulmonary system.

U.S. Pat. No. 5,543,158, incorporated herein by reference, providesbiodegradable injectable particles having a biodegradable solid corecontaining a biologically active material and poly(alkylene glycol)moieties on the surface.

WO2012135025 (also published as US20120251560), incorporated herein byreference, describes conjugated polyethyleneimine (PEI) polymers andconjugated aza-macrocycles (collectively referred to as “conjugatedlipomer” or “lipomers”). In certain embodiments, it can envisioned thatsuch conjugated lipomers can be used in the context of the CRISPR-Cassystem to achieve in vitro, ex vivo and in vivo genomic perturbations tomodify gene expression, including modulation of protein expression.

In one embodiment, the nanoparticle may be epoxide-modifiedlipid-polymer, advantageously 7C1 (see, e.g., James E. Dahlman andCarmen Barnes et al. Nature Nanotechnology (2014) published online 11May 2014, doi:10.1038/nnano.2014.84). C71 was synthesized by reactingC15 epoxide-terminated lipids with PEI600 at a 14:1 molar ratio, and wasformulated with C14PEG2000 to produce nanoparticles (diameter between 35and 60 nm) that were stable in PBS solution for at least 40 days.

An epoxide-modified lipid-polymer may be utilized to deliver theCRISPR-Cas system of the present invention to pulmonary, cardiovascularor renal cells, however, one of skill in the art may adapt the system todeliver to other target organs. Dosage ranging from about 0.05 to about0.6 mg/kg are envisioned. Dosages over several days or weeks are alsoenvisioned, with a total dosage of about 2 mg/kg.

In some embodiments, the LNP for delivering the RNA molecules isprepared by methods known in the art, such as those described in, forexample, WO 2005/105152 (PCT/EP2005/004920), WO 2006/069782(PCT/EP2005/014074), WO 2007/121947 (PCT/EP2007/003496), and WO2015/082080 (PCT/EP2014/003274), which are herein incorporated byreference. LNPs aimed specifically at the enhanced and improved deliveryof siRNA into mammalian cells are described in, for example, Aleku etal., Cancer Res., 68(23): 9788-98 (Dec. 1, 2008), Strumberg et al., Int.J. Clin. Pharmacol. Ther., 50(1): 76-8 (Jan. 2012), Schultheis et al.,J. Clin. Oncol., 32(36): 4141-48 (Dec. 20, 2014), and Fehring et al.,Mol. Ther., 22(4): 811-20 (Apr. 22, 2014), which are herein incorporatedby reference and may be applied to the present technology.

In some embodiments, the LNP includes any LNP disclosed in WO2005/105152 (PCT/EP2005/004920), WO 2006/069782 (PCT/EP2005/014074), WO2007/121947 (PCT/EP2007/003496), and WO 2015/082080 (PCT/EP2014/003274).

In some embodiments, the LNP includes at least one lipid having FormulaI:

wherein R1 and R2 are each and independently selected from the groupcomprising alkyl, n is any integer between 1 and 4, and R3 is an acylselected from the group comprising lysyl, ornithyl, 2,4-diaminobutyryl,histidyl and an acyl moiety according to Formula II:

wherein m is any integer from 1 to 3 and Y— is a pharmaceuticallyacceptable anion. In some embodiments, a lipid according to Formula Iincludes at least two asymmetric C atoms. In some embodiments,enantiomers of Formula I include, but are not limited to, R-R; S-S; R-Sand S-R enantiomer.

In some embodiments, R1 is lauryl and R2 is myristyl. In anotherembodiment, R1 is palmityl and R2 is oleyl. In some embodiments, m is 1or 2. In some embodiments, Y— is selected from halogenids, acetate ortrifluoroacetate.

In some embodiments, the LNP comprises one or more lipids select from:

arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyl-amidetrihydrochloride (Formula III)

arginyl-2,3-diamino propionic acid-N-lauryl-N-myristyl-amidetrihydrochloride (Formula IV)

arginyl-lysine-N-lauryl-N-myristyl-amide trihydrochloride (Formula V)

In some embodiments, the LNP also includes a constituent. By way ofexample, but not by way of limitation, in some embodiments, theconstituent is selected from peptides, proteins, oligonucleotides,polynucleotides, nucleic acids, or a combination thereof. In someembodiments, the constituent is an antibody, e.g., a monoclonalantibody. In some embodiments, the constituent is a nucleic acidselected from, e.g., ribozymes, aptamers, spiegelmers, DNA, RNA, PNA,LNA, or a combination thereof. In some embodiments, the nucleic acid isgRNA and/or mRNA.

In some embodiments, the constituent of the LNP comprises an mRNAencoding a CRIPSR effector protein. In some embodiments, the constituentof the LNP comprises an mRNA encoding a Type-II, Type-V, or Type-VICRIPSR effector protein. In some embodiments, the constituent of the LNPcomprises an mRNA encoding an RNA-guided DNA binding protein. In someembodiments, the constituent of the LNP comprises an mRNA encoding anRNA-guided RNA binding protein.

In some embodiments, the constituent of the LNP further comprises one ormore guide RNA. In some embodiments, the LNP is configured to deliverthe aforementioned mRNA and guide RNA to vascular endothelium. In someembodiments, the LNP is configured to deliver the aforementioned mRNAand guide RNA to pulmonary endothelium. In some embodiments, the LNP isconfigured to deliver the aforementioned mRNA and guide RNA to liver. Insome embodiments, the LNP is configured to deliver the aforementionedmRNA and guide RNA to lung. In some embodiments, the LNP is configuredto deliver the aforementioned mRNA and guide RNA to hearts. In someembodiments, the LNP is configured to deliver the aforementioned mRNAand guide RNA to spleen. In some embodiments, the LNP is configured todeliver the aforementioned mRNA and guide RNA to kidney. In someembodiments, the LNP is configured to deliver the aforementioned mRNAand guide RNA to pancrea. In some embodiments, the LNP is configured todeliver the aforementioned mRNA and guide RNA to brain. In someembodiments, the LNP is configured to deliver the aforementioned mRNAand guide RNA to macrophages.

In some embodiments, the LNP also includes at least one helper lipid. Insome embodiments, the helper lipid is selected from phospholipids andsteroids. In some embodiments, the phospholipids are di- and/ormonoester of the phosphoric acid. In some embodiments, the phospholipidsare phosphoglycerides and/or sphingolipids. In some embodiments, thesteroids are naturally occurring and/or synthetic compounds based on thepartially hydrogenated cyclopenta[a]phenanthrene. In some embodiments,the steroids contain 21 to 30 C atoms. In some embodiments, the steroidis cholesterol. In some embodiments, the helper lipid is selected from1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), ceramide, and1,2-dioleylsn-glycero-3-phosphoethanolamine (DOPE).

In some embodiments, the at least one helper lipid comprises a moietyselected from the group comprising a PEG moiety, a HEG moiety, apolyhydroxyethyl starch (polyHES) moiety and a polypropylene moiety. Insome embodiments, the moiety has a molecule weight between about 500 to10,000 Da or between about 2,000 to 5,000 Da. In some embodiments, thePEG moiety is selected from 1,2-distearoyl-sn-glycero-3phosphoethanolamine, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine, andCeramide-PEG. In some embodiments, the PEG moiety has a molecular weightbetween about 500 to 10,000 Da or between about 2,000 to 5,000 Da. Insome embodiments, the PEG moiety has a molecular weight of 2,000 Da.

In some embodiments, the helper lipid is between about 20 mol % to 80mol % of the total lipid content of the composition. In someembodiments, the helper lipid component is between about 35 mol % to 65mol % of the total lipid content of the LNP. In some embodiments, theLNP includes lipids at 50 mol % and the helper lipid at 50 mol % of thetotal lipid content of the LNP.

In some embodiments, the LNP includes any of-3-arginyl-2,3-diaminopropionic acid-N-palmityl-N-oleyl-amidetrihydrochloride, -arginyl-2,3-diaminopropionicacid-N-lauryl-N-myristyl-amide trihydrochloride orarginyl-lysine-N-lauryl-N-myristyl-amide trihydrochloride in combinationwith DPhyPE, wherein the content of DPhyPE is about 80 mol %, 65 mol %,50 mol % and 35 mol % of the overall lipid content of the LNP. In someembodiments, the LNP includes -arginyl-2,3-diamino propionicacid-N-pahnityl-N-oleyl-amide trihydrochloride (lipid) and1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (helper lipid). In someembodiments, the LNP includes -arginyl-2,3-diamino propionicacid-N-palmityl-N-oleyl-amide trihydrochloride (lipid),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (first helper lipid),and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-PEG2000 (secondhelper lipid).

In some embodiments, the second helper lipid is between about 0.05 mol %to 4.9 mol % or between about 1 mol % to 3 mol % of the total lipidcontent. In some embodiments, the LNP includes lipids at between about45 mol % to 50 mol % of the total lipid content, a first helper lipidbetween about 45 mol % to 50 mol % of the total lipid content, under theproviso that there is a PEGylated second helper lipid between about 0.1mol % to 5 mol %, between about 1 mol % to 4 mol %, or at about 2 mol %of the total lipid content, wherein the sum of the content of thelipids, the first helper lipid, and of the second helper lipid is 100mol % of the total lipid content and wherein the sum of the first helperlipid and the second helper lipid is 50 mol % of the total lipidcontent. In some embodiments, the LNP comprises: (a) 50 mol % of-arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyl-amidetrihydrochloride, 48 mol % of1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine; and 2 mol %1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000; or (b) 50 mol %of -arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyl-amidetrihydrocloride, 49 mol %1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine; and 1 mol %N(Carbonyl-methoxypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero3-phosphoethanolamine,or a sodium salt thereof.

In some embodiments, the LNP contains a nucleic acid, wherein the chargeratio of nucleic acid backbone phosphates to cationic lipid nitrogenatoms is about 1:1.5-7 or about 1:4.

In some embodiments, the LNP also includes a shielding compound, whichis removable from the lipid composition under in vivo conditions. Insome embodiments, the shielding compound is a biologically inertcompound. In some embodiments, the shielding compound does not carry anycharge on its surface or on the molecule as such. In some embodiments,the shielding compounds are polyethylenglycoles (PEGs),hydroxyethylglucose (HEG) based polymers, polyhydroxyethyl starch(polyHES) and polypropylene. In some embodiments, the PEG, HEG, polyHES,and a polypropylene weight between about 500 to 10,000 Da or betweenabout 2000 to 5000 Da. In some embodiments, the shielding compound isPEG2000 or PEG5000.

In some embodiments, the LNP includes at least one lipid, a first helperlipid, and a shielding compound that is removable from the lipidcomposition under in vivo conditions. In some embodiments, the LNP alsoincludes a second helper lipid. In some embodiments, the first helperlipid is ceramide. In some embodiments, the second helper lipid isceramide. In some embodiments, the ceramide comprises at least one shortcarbon chain substituent of from 6 to 10 carbon atoms. In someembodiments, the ceramide comprises 8 carbon atoms. In some embodiments,the shielding compound is attached to a ceramide. In some embodiments,the shielding compound is attached to a ceramide. In some embodiments,the shielding compound is covalently attached to the ceramide. In someembodiments, the shielding compound is attached to a nucleic acid in theLNP. In some embodiments, the shielding compound is covalently attachedto the nucleic acid. In some embodiments, the shielding compound isattached to the nucleic acid by a linker. In some embodiments, thelinker is cleaved under physiological conditions. In some embodiments,the linker is selected from ssRNA, ssDNA, dsRNA, dsDNA, peptide,S-S-linkers and pH sensitive linkers. In some embodiments, the linkermoiety is attached to the 3′ end of the sense strand of the nucleicacid. In some embodiments, the shielding compound comprises apH-sensitive linker or a pH-sensitive moiety. In some embodiments, thepH-sensitive linker or pH-sensitive moiety is an anionic linker or ananionic moiety. In some embodiments, the anionic linker or anionicmoiety is less anionic or neutral in an acidic environment. In someembodiments, the pH-sensitive linker or the pH-sensitive moiety isselected from the oligo (glutamic acid), oligophenolate(s) anddiethylene triamine penta acetic acid.

In any of the LNP embodiments in the previous paragraph, the LNP canhave an osmolality between about 50 to 600 mosmole/kg, between about 250to 350 mosmole/kg, or between about 280 to 320 mosmole/kg, and/orwherein the LNP formed by the lipid and/or one or two helper lipids andthe shielding compound have a particle size between about 20 to 200 nm,between about 30 to 100 nm, or between about 40 to 80 nm.

In some embodiments, the shielding compound provides for a longercirculation time in vivo and allows for a better biodistribution of thenucleic acid containing LNP. In some embodiments, the shielding compoundprevents immediate interaction of the LNP with serum compounds orcompounds of other bodily fluids or cytoplasma membranes, e.g.,cytoplasma membranes of the endothelial lining of the vasculature, intowhich the LNP is administered. Additionally or alternatively, in someembodiments, the shielding compounds also prevent elements of the immunesystem from immediately interacting with the LNP. Additionally oralternatively, in some embodiments, the shielding compound acts as ananti-opsonizing compound. Without wishing to be bound by any mechanismor theory, in some embodiments, the shielding compound forms a cover orcoat that reduces the surface area of the LNP available for interactionwith its environment. Additionally or alternatively, in someembodiments, the shielding compound shields the overall charge of theLNP.

In another embodiment, the LNP includes at least one cationic lipidhaving Formula VI:

wherein n is 1, 2, 3, or 4, wherein m is 1, 2, or 3, wherein Y— isanion, wherein each of R1 and R2 is individually and independentlyselected from the group consisting of linear C12-C18 alkyl and linearC12-C18 alkenyl, a sterol compound, wherein the sterol compound isselected from the group consisting of cholesterol and stigmasterol, anda PEGylated lipid, wherein the PEGylated lipid comprises a PEG moiety,wherein the PEGylated lipid is selected from the group consisting of:a PEGylated phosphoethanolamine of Formula VII:

wherein R3 and R4 are individually and independently linear C13-C17alkyl, and p is any integer between 15 to 130;a PEGylated ceramide of Formula VIII:

wherein R5 is linear C7-C15 alkyl, and q is any number between 15 to130; anda PEGylated diacylglycerol of Formula IX:

wherein each of R6 and R7 is individually and independently linearC11-C17 alkyl, and r is any integer from 15 to 130.

In some embodiments, R1 and R2 are different from each other. In someembodiments, R1 is palmityl and R2 is oleyl. In some embodiments, R1 islauryl and R2 is myristyl. In some embodiments, R1 and R2 are the same.In some embodiments, each of R1 and R2 is individually and independentlyselected from the group consisting of C12 alkyl, C14 alkyl, C16 alkyl,C18 alkyl, C12 alkenyl, C14 alkenyl, C16 alkenyl and C18 alkenyl. Insome embodiments, each of C12 alkenyl, C14 alkenyl, C16 alkenyl and C1 8alkenyl comprises one or two double bonds. In some embodiments, C18alkenyl is C18 alkenyl with one double bond between C9 and C10. In someembodiments, C18 alkenyl is cis-9-octadecyl.

In some embodiments, the cationic lipid is a compound of Formula X:

In some embodiments, Y— is selected from halogenids, acetate andtrifluoroacetate. In some embodiments, the cationic lipid is-arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyl-amidetrihydrochloride of Formula III:

In some embodiments, the cationic lipid is -arginyl-2,3-diaminopropionic acid-N-lauryl-N-myristyl-amide trihydrochloride of Formula IV:

In some embodiments, the cationic lipid is-arginyl-lysine-N-lauryl-N-myristyl-amide trihydrochloride of Formula V:

In some embodiments, the sterol compound is cholesterol. In someembodiments, the sterol compound is stigmasterin.

In some embodiments, the PEG moiety of the PEGylated lipid has amolecular weight from about 800 to 5,000 Da. In some embodiments, themolecular weight of the PEG moiety of the PEGylated lipid is about 800Da. In some embodiments, the molecular weight of the PEG moiety of thePEGylated lipid is about 2,000 Da. In some embodiments, the molecularweight of the PEG moiety of the PEGylated lipid is about 5,000 Da. Insome embodiments, the PEGylated lipid is a PEGylated phosphoethanolamineof Formula VII, wherein each of R3 and R4 is individually andindependently linear C13-C17 alkyl, and p is any integer from 18, 19 or20, or from 44, 45 or 46 or from 113, 114 or 115. In some embodiments,R3 and R4 are the same. In some embodiments, R3 and R4 are different. Insome embodiments, each of R3 and R4 is individually and independentlyselected from the group consisting of C13 alkyl, C15 alkyl and C17alkyl. In some embodiments, the PEGylated phosphoethanolamine of FormulaVII is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N—[methoxy(polyethyleneglycol)-2000](ammonium salt):

In some embodiments, the PEGylated phosphoethanolamine of Formula VII is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N—[methoxy(polyethyleneglycol)-5000](ammonium salt):

In some embodiments, the PEGylated lipid is a PEGylated ceramide ofFormula VIII, wherein R5 is linear C7-C15 alkyl, and q is any integerfrom 18, 19 or 20, or from 44, 45 or 46 or from 113, 114 or 115. In someembodiments, R5 is linear C7 alkyl. In some embodiments, R5 is linearC15 alkyl. In some embodiments, the PEGylated ceramide of Formula VIIIis N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethyleneglycol)2000]}:

In some embodiments, the PEGylated ceramide of Formula VIII isN-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}

In some embodiments, the PEGylated lipid is a PEGylated diacylglycerolof Formula IX, wherein each of R6 and R7 is individually andindependently linear C11-C17 alkyl, and r is any integer from 18, 19 or20, or from 44, 45 or 46 or from 113, 114 or 115. In some embodiments,R6 and R7 are the same. In some embodiments, R6 and R7 are different. Insome embodiments, each of R6 and R7 is individually and independentlyselected from the group consisting of linear C17 alkyl, linear C15 alkyland linear C13 alkyl. In some embodiments, the PEGylated diacylglycerolof Formula IX 1,2-Distearoyl-sn-glycerol [methoxy(polyethyleneglycol)2000]:

In some embodiments, the PEGylated diacylglycerol of Formula IX is1,2-Dipalmitoyl-sn-glycerol [methoxy(polyethylene glycol)2000]:

In some embodiments, the PEGylated diacylglycerol of Formula IX is:

In some embodiments, the LNP includes at least one cationic lipidselected from of Formulas III, IV, and V, at least one sterol compoundselected from a cholesterol and stigmasterin, and wherein the PEGylatedlipid is at least one selected from Formulas XI and XII. In someembodiments, the LNP includes at least one cationic lipid selected fromFormulas III, IV, and V, at least one sterol compound selected from acholesterol and stigmasterin, and wherein the PEGylated lipid is atleast one selected from Formulas XIII and XIV. In some embodiments, theLNP includes at least one cationic lipid selected from Formulas III, IV,and V, at least one sterol compound selected from a cholesterol andstigmasterin, and wherein the PEGylated lipid is at least one selectedfrom Formulas XV and XVI. In some embodiments, the LNP includes acationic lipid of Formula III, a cholesterol as the sterol compound, andwherein the PEGylated lipid is Formula XI.

In any of the LNP embodiments in the previous paragraph, wherein thecontent of the cationic lipid composition is between about 65 mole % to75 mole %, the content of the sterol compound is between about 24 mole %to 34 mole % and the content of the PEGylated lipid is between about 0.5mole % to 1.5 mole %, wherein the sum of the content of the cationiclipid, of the sterol compound and of the PEGylated lipid for the lipidcomposition is 100 mole %. In some embodiments, the cationic lipid isabout 70 mole %, the content of the sterol compound is about 29 mole %and the content of the PEGylated lipid is about 1 mole %. In someembodiments, the LNP is 70 mole % of Formula III, 29 mole % ofcholesterol, and 1 mole % of Formula XI.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (MHC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by nanoparticle tracking analysis (NTA)and electron microscopy. Alvarez-Erviti et al. obtained 6-12 μg ofexosomes (measured based on protein concentration) per 106 cells.

Next, Alvarez-Erviti et al. investigated the possibility of loadingmodified exosomes with exogenous cargoes using electroporation protocolsadapted for nanoscale applications. As electroporation for membraneparticles at the nanometer scale is not well-characterized, nonspecificCy5-labeled RNA was used for the empirical optimization of theelectroporation protocol. The amount of encapsulated RNA was assayedafter ultracentrifugation and lysis of exosomes. Electroporation at 400V and 125 μF resulted in the greatest retention of RNA and was used forall subsequent experiments.

Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNAencapsulated in 150 μg of RVG exosomes to normal C57BL/6 mice andcompared the knockdown efficiency to four controls: untreated mice, miceinjected with RVG exosomes only, mice injected with BACE1 siRNAcomplexed to an in vivo cationic liposome reagent and mice injected withBACE1 siRNA complexed to RVG-9R, the RVG peptide conjugated to 9D-arginines that electrostatically binds to the siRNA. Cortical tissuesamples were analyzed 3 d after administration and a significant proteinknockdown (45%, P<0.05, versus 62%, P<0.01) in both siRNA-RVG-9R-treatedand siRNARVG exosome-treated mice was observed, resulting from asignificant decrease in BACE1 mRNA levels (66% [+ or −] 15%, P<0.001 and61% [+ or −] 13% respectively, P<0.01). Moreover, Applicantsdemonstrated a significant decrease (55%, P<0.05) in the total[beta]-amyloid 1-42 levels, a main component of the amyloid plaques inAlzheimer's pathology, in the RVG-exosome-treated animals. The decreaseobserved was greater than the β-amyloid 1-40 decrease demonstrated innormal mice after intraventricular injection of BACE1 inhibitors.Alvarez-Erviti et al. carried out 5′-rapid amplification of cDNA ends(RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA.

Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomesinduced immune responses in vivo by assessing IL-6, IP-10, TNFα andIFN—α serum concentrations. Following exosome treatment, nonsignificantchanges in all cytokines were registered similar to siRNA-transfectionreagent treatment in contrast to siRNA-RVG-9R, which potently stimulatedIL-6 secretion, confirming the immunologically inert profile of theexosome treatment. Given that exosomes encapsulate only 20% of siRNA,delivery with RVG-exosome appears to be more efficient than RVG-9Rdelivery as comparable mRNA knockdown and greater protein knockdown wasachieved with fivefold less siRNA without the corresponding level ofimmune stimulation. This experiment demonstrated the therapeuticpotential of RVG-exosome technology, which is potentially suited forlong-term silencing of genes related to neurodegenerative diseases. Theexosome delivery system of Alvarez-Erviti et al. may be applied todeliver the CRISPR-Cas system of the present invention to therapeutictargets, especially neurodegenerative diseases. A dosage of about 100 to1000 mg of CRISPR Cas encapsulated in about 100 to 1000 mg of RVGexosomes may be contemplated for the present invention.

El-Andaloussi et al. (Nature Protocols 7,2112-2126(2012)) discloses howexosomes derived from cultured cells can be harnessed for delivery ofRNA in vitro and in vivo. This protocol first describes the generationof targeted exosomes through transfection of an expression vector,comprising an exosomal protein fused with a peptide ligand. Next,El-Andaloussi et al. explain how to purify and characterize exosomesfrom transfected cell supernatant. Next, El-Andaloussi et al. detailcrucial steps for loading RNA into exosomes. Finally, El-Andaloussi etal. outline how to use exosomes to efficiently deliver RNA in vitro andin vivo in mouse brain. Examples of anticipated results in whichexosome-mediated RNA delivery is evaluated by functional assays andimaging are also provided. The entire protocol takes ˜3 weeks. Deliveryor administration according to the invention may be performed usingexosomes produced from self-derived dendritic cells. From the hereinteachings, this can be employed in the practice of the invention.

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention.

Exosomes from plasma can be prepared by centrifugation of buffy coat at900 g for 20 min to isolate the plasma followed by harvesting cellsupernatants, centrifuging at 300 g for 10 min to eliminate cells and at16 500 g for 30 min followed by filtration through a 0.22 mm filter.Exosomes are pelleted by ultracentrifugation at 120 000 g for 70 min.Chemical transfection of siRNA into exosomes is carried out according tothe manufacturer's instructions in RNAi Human/Mouse Starter Kit(Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a finalconcentration of 2 mmol/ml. After adding HiPerFect transfection reagent,the mixture is incubated for 10 min at RT. In order to remove the excessof micelles, the exosomes are re-isolated using aldehyde/sulfate latexbeads. The chemical transfection of CRISPR Cas into exosomes may beconducted similarly to siRNA. The exosomes may be co-cultured withmonocytes and lymphocytes isolated from the peripheral blood of healthydonors. Therefore, it may be contemplated that exosomes containingCRISPR Cas may be introduced to monocytes and lymphocytes of andautologously reintroduced into a human. Accordingly, delivery oradministration according to the invention may be performed using plasmaexosomes.

Liposomes

The lipid, lipid particle, or lipid bylayer or lipid entity of theinvention can be prepared by methods well known in the art. See Wang etal., ACS Synthetic Biology, 1, 403-07 (2012); Wang et al., PNAS, 113(11)2868-2873 (2016); Manoharan, et al., WO 2008/042973; Zugates et al.,U.S. Pat. No. 8,071,082; Xu et al., WO 2014/186366 A1 (US20160082126).Xu et provides a way to make a nanocomplex for the delivery of saporinwherein the nanocomplex comprising saporin and a lipid-like compound,and wherein the nanocomplex has a particle size of 50 nm to 1000 nm; thesaporin binds to the lipid-like compound via non-covalent interaction orcovalent bonding; and the lipid-like compound has a hydrophilic moiety,a hydrophobic moiety, and a linker joining the hydrophilic moiety andthe hydrophobic moiety, the hydrophilic moiety being optionally chargedand the hydrophobic moiety having 8 to 24 carbon atoms. Xu et al., WO2014/186348 (US20160129120) provides examples of nanocomplexes ofmodified peptides or proteins comprising a cationic delivery agent andan anionic pharmaceutical agent, wherein the nanocomplex has a particlesize of 50 to 1000 nm, the cationic delivery agent binds to the anionicpharmaceutical agent, and the anionic pharmaceutical agent is a modifiedpeptide or protein formed of a peptide and a protein and an addedchemical moiety that contains an anionic group. The added chemicalmoiety is linked to the peptide or protein via an amide group, an estergroup, an ether group, a thioether group, a disulfide group, a hydrazonegroup, a sulfenate ester group, an amidine group, a urea group, acarbamate group, an imidoester group, or a carbonate group. Moreparticularly these documents provide examples of lipid or lipid-likecompounds that can be used to make the particle delivery system of thepresent invention, including compounds of the formula B1-K1-A-K2-B2, inwhich A, the hydrophilic moiety, is

each of Ra, Ra′, Ra″, and Ra′″, independently, being a C1-C20 monovalentaliphatic radical, a C1-C20 0 monovalent heteroaliphatic radical, amonovalent aryl radical, or a monovalent heteroaryl radical; and Z beinga C1-C20 bivalent aliphatic radical, a C1-C20 bivalent heteroaliphaticradical, a bivalent aryl radical, or a bivalent heteroaryl radical; eachof Bi, the hydrophobic moiety, and B2, also the hydrophobic moiety,independently, is a C12-20 aliphatic radical or a C12-20 heteroaliphaticradical; and each of K1, the linker, and K2, also the linker,independently, is O, S, Si, C1-C6 alkylene

in which each of m, n, p, q, and t, independently, is 1-6; W is O, S, orNRC; each of L1, L3, L5, L7, and L9, independently, is a bond, O, S, orNRd; each of L2, L4, L6, L8, and L10, independently, is a bond, O, S, orNRe; and V is ORf, SRg, or NRhRi, each of Rb, Rc, Rd, Re, Rf, Rg, Rh,and Ri, independently, being H, OH, a C1-C10 oxyaliphatic radical, aC1-C10 monovalent aliphatic radical, a C1-C10 monovalent heteroaliphaticradical, a monovalent aryl radical, or a monovalent heteroaryl radicaland specific compounds:

Additional examples of cationic lipid that can be used to make theparticle delivery system of the invention can be found in US20150140070,wherein the cationic lipid has the formula

wherein p is an integer between 1 and 9, inclusive; each instance of Qis independently O, S, or NRQ; RQ is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of the formula (i), (ii) or (iii);each instance of R1 is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, halogen,—ORA1, —N(RA1)2, -SRA1, or a group of formula:

L is an optionally substituted alkylene, optionally substitutedalkenylene, optionally substituted alkynylene, optionally substitutedheteroalkylene, optionally substituted heteroalkenylene, optionallysubstituted heteroalkynylene, optionally substituted carbocyclylene,optionally substituted heterocyclylene, optionally substituted arylene,or optionally substituted heteroarylene, or combination thereof, andeach of R6 and R7 is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of formula (i), (ii) or (iii);each occurrence of RA1 is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to an oxygen atom, a sulfurprotecting group when attached to an sulfur atom, a nitrogen protectinggroup when attached to a nitrogen atom, or two RA1 groups, together withthe nitrogen atom to which they are attached, are joined to form anoptionally substituted heterocyclic or optionally substituted heteroarylring; each instance of R2 is independently hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, a nitrogen protecting group, or a group of theformula (i), (ii), or (iii); Formulae (i), (ii), and (iii) are:

each instance of R′ is independently hydrogen or optionally substitutedalkyl; X is O, S, or NRX; RX is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group; Y is O, S, or NRY; RY is hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, or a nitrogen protecting group; RP is hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, an oxygen protecting group when attached to anoxygen atom, a sulfur protecting group when attached to a sulfur atom,or a nitrogen protecting group when attached to a nitrogen atom; RL isoptionally substituted C1-50 alkyl, optionally substituted C2-50alkenyl, optionally substituted C2-50 alkynyl, optionally substitutedheteroC1-50 alkyl, optionally substituted heteroC2-50 alkenyl,optionally substituted heteroC2-50 alkynyl, or a polymer; provided thatat least one instance of RQ, R2, R6, or R7 is a group of the formula(i), (ii), or (iii); in Liu et al., (US 20160200779, US 20150118216, US20150071903, and US 20150071903), which provide examples of cationiclipids to include polyethylenimine, polyamidoamine (PAMAM) starburstdendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase,LIPOFECTAMINE® (e.g., LIPOFECTAMINE® 2000, LIPOFECTAMINE® 3000,LIPOFECTAMINE® RNAiMAX, LIPOFECTAMINE® LTX), SAINT-RED (SynvoluxTherapeutics, Groningen Netherlands), DOPE, Cytofectin (Gilead Sciences,Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.).Exemplary cationic liposomes can be made fromN—[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA),N—[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate(DOTAP), 3.beta.-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol(DC-Chol),2,3,-dioleyloxy-N—[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamin-ium trifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; anddimethyldioctadecylammonium bromide (DDAB); in WO2013/093648 whichprovides cationic lipids of formula

in which Z=an alkyl linker, C2-C4 alkyl, Y=an alkyl linker, C1-C6 alkyl,R1 and R2 are each independently C10-C30alkyl, C10-C30alkenyl, orC10-C30alkynyl, C10-C30alkyl, C10-C20alkyl, C12-C18alkyl, C13-C17alkyl,C13alkyl, C10-C30alkenyl, C10-C20alkenyl. C12-C18alkenyl,C13-C17alkenyl, C17alkenyl; R3 and R4 are each independently hydrogen,C1-C6 alkyl, or —CH2CH2OH, C1-C6 alkyl, C1-C3alkyl; n is 1-6; and X is acounterion, including any nitrogen counterion, as that term is readilyunderstood in the art, and specific cationic lipids including

WO2013/093648 also provides examples of other cationic charged lipids atphysiological pH including N,N-dioleyl-N,N-dimethylammonium chloride(DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE) and dioctadecylamidoglycyl carboxyspermidine (DOGS);inUS 20160257951, which provides cationic lipids with a general formula

or a pharmacologically acceptable salt thereof, wherein R1 and R2 areeach independently a hydrogen atom, a C1-C6 alkyl group optionallysubstituted with one or more substituents selected from substituentgroup a, a C2-C6 alkenyl group optionally substituted with one or moresubstituents selected from substituent group a, a C2-C6 alkynyl groupoptionally substituted with one or more substituents selected fromsubstituent group a, or a C3-C7 cycloalkyl group optionally substitutedwith one or more substituents selected from substituent group a, or R1and R2 form a 3- to 10-membered heterocyclic ring together with thenitrogen atom bonded thereto, wherein the heterocyclic ring isoptionally substituted with one or more substituents selected fromsubstituent group a and optionally contains one or more atoms selectedfrom a nitrogen atom, an oxygen atom, and a sulfur atom, in addition tothe nitrogen atom bonded to Ri and R2, as atoms constituting theheterocyclic ring; R8 is a hydrogen atom or a C1-C6 alkyl groupoptionally substituted with one or more substituents selected fromsubstituent group a; or R1 and R8 together are the group -(CH2)q-;substituent group a consists of a halogen atom, an oxo group, a hydroxygroup, a sulfanyl group, an amino group, a cyano group, a C1-C6 alkylgroup, a C1-C6 halogenated alkyl group, a C1-C6 alkoxy group, a C1-C6alkylsulfanyl group, a C1-C6 alkylamino group, and a C1-C7 alkanoylgroup; L1 is a C10-C24 alkyl group optionally substituted with one ormore substituents selected from substituent group β1, a C10-C24 alkenylgroup optionally substituted with one or more substituents selected fromsubstituent group β1, a C3-C24 alkynyl group optionally substituted withone or more substituents selected from substituent group β1, or a(C1-C10 alkyl)-(Q)k-(C1-C10 alkyl) group optionally substituted with oneor more substituents selected from substituent group β1; L2 is,independently of L1, a C10-C24 alkyl group optionally substituted withone or more substituents selected from substituent group β1, a C10-C24alkenyl group optionally substituted with one or more substituentsselected from substituent group β1, a C3-C24 alkynyl group optionallysubstituted with one or more substituents selected from substituentgroup β1, a (C1-C10 alkyl)-(Q)k-(C1-C10 alkyl) group optionallysubstituted with having one or more substituents selected fromsubstituent group β1, a (C10-C24 alkoxy)methyl group optionallysubstituted with one or more substituents selected from substituentgroup β1, a (C10-C24 alkenyl)oxymethyl group optionally substituted withone or more substituents selected from substituent group β1, a (C3-C24alkynyl)oxymethyl group optionally substituted with one or moresubstituents selected from substituent group p31, or a (C1-C10alkyl)-(Q)k-(C1-C10 alkoxy)methyl group optionally substituted with oneor more substituents selected from substituent group β1; substituentgroup p31 consists of a halogen atom, an oxo group, a cyano group, aC1-C6 alkyl group, a C1-C6 halogenated alkyl group, a C1-C6 alkoxygroup, a C1-C6 alkylsulfanyl group, a C1-C7 alkanoyl group, a C1-C7alkanoyloxy group, a C3-C7 alkoxyalkoxy group, a (C1-C6 alkoxy)carbonylgroup, a (C1-C6 alkoxy)carboxyl group, a (C1-C6 alkoxy)carbamoyl group,and a (C1-C6 alkylamino)carboxyl group; Q is a group of formula:

when L1 and L2 are each substituted with one or more substituentsselected from substituent group β1 and substituent group β1 is a C1-C6alkyl group, a C1-C6 alkoxy group, a C1-C6 alkylsulfanyl group, a C1-C7alkanoyl group, or a C1-C7 alkanoyloxy group, the substituent orsubstituents selected from substituent group β1 in L1 and thesubstituent or substituents selected from substituent group β1 in L2optionally bind to each other to form a cyclic structure; k is 1, 2, 3,4, 5, 6, or 7; m is 0 or 1; p is 0, 1, or 2; q is 1, 2, 3, or 4; and ris 0, 1, 2, or 3, provided that p+r is 2 or larger, or q+r is 2 orlarger, and specific cationic lipids including

and in US 20160244761, which provides cationic lipids that include1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA),1,2-di-.gamma.-linolenyloxy-N,N-dimethylaminopropane (.gamma.-DLenDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLin-K-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA)(also known as DLin-C2K-DMA, XTC2, and C2K),2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane(DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane(DLin-K-C4-DMA),1,2-dilinolenyloxy-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLen-C2K-DMA),1,2-di-.gamma.-linolenyloxy-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(.gamma.-DLen-C2K-DMA), dilinoleylmethyl-3-dimethylaminopropionate(DLin-M-C2-DMA) (also known as MC2),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA) (also known as MC3) and3-(dilinoleylmethoxy)-N,N-dimethylpropan-1-amine (DLin-MP-DMA) (alsoknown as 1-B1 1).

In one embodiment, the lipid compound is preferably a bio-reduciblematerial, e.g., a bio-reducible polymer and a bio-reducible lipid-likecompound.

In embodiment, the lipid compound comprises a hydrophilic head, and ahydrophobic tail, and optionally a linker.

In one embodiment, the hydrophilic head contains one or more hydrophilicfunctional groups, e.g., hydroxyl, carboxyl, amino, sulfhydryl,phosphate, amide, ester, ether, carbamate, carbonate, carbamide andphosphodiester. These groups can form hydrogen bonds and are optionallypositively or negatively charged, in particular at physiologicalconditions such as physiological pH.

In one embodiment, the hydrophobic tail is a saturated or unsaturated,linear or branched, acyclic or cyclic, aromatic or nonaromatichydrocarbon moiety, wherein the saturated or unsaturated, linear orbranched, acyclic or cyclic, aromatic or nonaromatic hydrocarbon moietyoptionally contains a disulfide bond and/or 8-24 carbon atoms. One ormore of the carbon atoms can be replaced with a heteroatom, such as N,O, P, B, S, Si, Sb, A1, Sn, As, Se, and Ge. The lipid or lipid-likecompounds containing disulfide bond can be bioreducible.

In one embodiment, the linker of the lipid or lipid-like compound linksthe hydrophilic head and the hydrophobic tail. The linker can be anychemical group that is hydrophilic or hydrophobic, polar or non-polar,e.g., O, S, Si, amino, alkylene, ester, amide, carbamate, carbamide,carbonate phosphate, phosphite, sulfate, sulfite, and thiosulfate.

The lipid or lipid-like compounds described above include the compoundsthemselves, as well as their salts and solvates, if applicable. A salt,for example, can be formed between an anion and a positively chargedgroup (e.g., amino) on a lipid-like compound. Suitable anions includechloride, bromide, iodide, sulfate, nitrate, phosphate, citrate,methanesulfonate, trifluoroacetate, acetate, malate, tosylate, tartrate,fumurate, glutamate, glucuronate, lactate, glutarate, and maleate.Likewise, a salt can also be formed between a cation and a negativelycharged group (e.g., carboxylate) on a lipid-like compound. Suitablecations include sodium ion, potassium ion, magnesium ion, calcium ion,and an ammonium cation such as tetramethylammonium ion. The lipid-likecompounds also include those salts containing quaternary nitrogen atoms.A solvate refers to a complex formed between a lipid-like compound and apharmaceutically acceptable solvent. Examples of pharmaceuticallyacceptable solvents include water, ethanol, isopropanol, ethyl acetate,acetic acid, and ethanolamine.

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review).

Liposomes can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes as drugcarriers. Although liposome formation is spontaneous when a lipid filmis mixed with an aqueous solution, it can also be expedited by applyingforce in the form of shaking by using a homogenizer, sonicator, or anextrusion apparatus (see, e.g., Spuch and Navarro, Journal of DrugDelivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

A liposome formulation may be mainly comprised of natural phospholipidsand lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline(DSPC), sphingomyelin, egg phosphatidylcholines andmonosialoganglioside. Since this formulation is made up of phospholipidsonly, liposomal formulations have encountered many challenges, one ofthe ones being the instability in plasma. Several attempts to overcomethese challenges have been made, specifically in the manipulation of thelipid membrane. One of these attempts focused on the manipulation ofcholesterol. Addition of cholesterol to conventional formulationsreduces rapid release of the encapsulated bioactive compound into theplasma or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increasesthe stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

In a particularly advantageous embodiment, Trojan Horse liposomes (alsoknown as Molecular Trojan Horses) are desirable and protocols may befound at http://cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long.These particles allow delivery of a transgene to the entire brain afteran intravascular injection. Without being bound by limitation, it isbelieved that neutral lipid particles with specific antibodiesconjugated to surface allow crossing of the blood brain barrier viaendocytosis. Applicant postulates utilizing Trojan Horse Liposomes todeliver the CRISPR family of nucleases to the brain via an intravascularinjection, which would allow whole brain transgenic animals without theneed for embryonic manipulation. About 1-5 g of DNA or RNA may becontemplated for in vivo administration in liposomes.

In another embodiment, the CRISPR Cas system or components thereof maybe administered in liposomes, such as a stable nucleic-acid-lipidparticle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology,Vol. 23, No. 8, August 2005). Daily intravenous injections of about 1, 3or 5 mg/kg/day of a specific CRISPR Cas targeted in a SNALP arecontemplated. The daily treatment may be over about three days and thenweekly for about five weeks. In another embodiment, a specific CRISPRCas encapsulated SNALP) administered by intravenous injection to atdoses of about 1 or 2.5 mg/kg are also contemplated (see, e.g.,Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006). The SNALPformulation may contain the lipids 3-N—[(wmethoxypoly(ethylene glycol)2000) carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic-acid-lipid particles (SNALPs) haveproven to be effective delivery molecules to highly vascularizedHepG2-derived liver tumors but not in poorly vascularized HCT-116derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780).The SNALP liposomes may be prepared by formulating D-Lin-DMA andPEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol andsiRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio ofCholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes areabout 80-100 nm in size.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine(Avanti Polar Lipids, Alabaster, Ala., USA), 3-N—[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, andcationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g.,Geisbert et al., Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kgtotal CRISPR Cas per dose administered as, for example, a bolusintravenous infusion may be contemplated.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC;Avanti Polar Lipids Inc.), PEG-cDMA, and1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g.,Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for invivo studies may comprise a final lipid/RNA mass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids—an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties.

To date, two clinical programs have been initiated using SNALPformulations with RNA. Tekmira Pharmaceuticals recently completed aphase I single-dose study of SNALP-ApoB in adult volunteers withelevated LDL cholesterol. ApoB is predominantly expressed in the liverand jejunum and is essential for the assembly and secretion of VLDL andLDL. Seventeen subjects received a single dose of SNALP-ApoB (doseescalation across 7 dose levels). There was no evidence of livertoxicity (anticipated as the potential dose-limiting toxicity based onpreclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial.

Alnylam Pharmaceuticals has similarly advanced ALN-TTR01, which employsthe SNALP technology described above and targets hepatocyte productionof both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). ThreeATTR syndromes have been described: familial amyloidotic polyneuropathy(FAP) and familial amyloidotic cardiomyopathy (FAC) both caused byautosomal dominant mutations in TTR; and senile systemic amyloidosis(SSA) cause by wildtype TTR. A placebo-controlled, singledose-escalation phase I trial of ALN-TTR01 was recently completed inpatients with ATTR. ALN-TTR01 was administered as a 15-minute IVinfusion to 31 patients (23 with study drug and 8 with placebo) within adose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatment was welltolerated with no significant increases in liver function tests.Infusion-related reactions were noted in 3 of 23 patients at ≥0.4 mg/kg;all responded to slowing of the infusion rate and all continued onstudy. Minimal and transient elevations of serum cytokines IL-6, IP-10and IL-Ira were noted in two patients at the highest dose of 1 mg/kg (asanticipated from preclinical and NHP studies). Lowering of serum TTR,the expected pharmacodynamics effect of ALN-TTR01, was observed at 1mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Niotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177). Thelipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) withmixing to a final ethanol and lipid concentration of 30% (vol/vol) and6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Northern Lipids) until a vesicle diameter of 70-90 nm, as determined bydynamic light scattering analysis, was obtained. This generally required1-3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueoussolution containing 30% ethanol) was added to the pre-equilibrated (35°C.) vesicles at a rate of ˜5 ml/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubatedfor a further 30 min at 35° C. to allow vesicle reorganization andencapsulation of the siRNA. The ethanol was then removed and theexternal buffer replaced with PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mMKH2PO4, pH 7.5) by either dialysis or tangential flow diafiltration.siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti PolarLipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molarratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles,SNALP were dialyzed against PBS and filter sterilized through a 0.2 μmfilter before use. Mean particle sizes were 75-85 nm and 90-95% of thesiRNA was encapsulated within the lipid particles. The final siRNA/lipidratio in formulations used for in vivo testing was ˜0.15 (wt/wt).LNP-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile PBS immediately before use and theformulations were administered intravenously through the lateral tailvein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the CRISPR Cas system of the presentinvention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate CRISPR Cas or components thereof or nucleicacid molecule(s) coding therefor e.g., similar to SiRNA (see, e.g.,Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533), and hence may beemployed in the practice of the invention. A preformed vesicle with thefollowing lipid composition may be contemplated: amino lipid,distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11+0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the guide RNA. Particles containing the highly potentamino lipid 16 may be used, in which the molar ratio of the four lipidcomponents 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) whichmay be further optimized to enhance in vivo activity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume:29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with the CRISPR Cassystem of the present invention or component(s) thereof or nucleic acidmolecule(s) coding therefor to form lipid nanoparticles (LNPs). Lipidsinclude, but are not limited to, DLin-KC2-DMA4, C12-200 and colipidsdisteroylphosphatidyl choline, cholesterol, and PEG-DMG may beformulated with CRISPR Cas instead of siRNA (see, e.g., Novobrantseva,Molecular Therapy-Nucleic Acids (2012) 1, e4; doi:10.1038/mtna.2011.3)using a spontaneous vesicle formation procedure. The component molarratio may be about 50/10/38.5/1.5 (DLin-KC2-DMA orC12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG). The finallipid:siRNA weight ratio may be ˜12:1 and 9:1 in the case ofDLin-KC2-DMA and C12-200 lipid nanoparticles (LNPs), respectively. Theformulations may have mean particle diameters of ˜80 nm with >90%entrapment efficiency. A 3 mg/kg dose may be contemplated.

Tekmira has a portfolio of approximately 95 patent families, in the U.S.and abroad, that are directed to various aspects of LNPs and LNPformulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069;8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263;7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos 1766035;1519714; 1781593 and 1664316), all of which may be used and/or adaptedto the present invention.

The CRISPR Cas system or components thereof or nucleic acid molecule(s)coding therefor may be delivered encapsulated in PLGA Microspheres suchas that further described in US published applications 20130252281 and20130245107 and 20130244279 (assigned to Moderna Therapeutics) whichrelate to aspects of formulation of compositions comprising modifiednucleic acid molecules which may encode a protein, a protein precursor,or a partially or fully processed form of the protein or a proteinprecursor. The formulation may have a molar ratio 50:10:38.5:1.5-3.0(cationic lipid:fusogenic lipid:cholesterol:PEG lipid). The PEG lipidmay be selected from, but is not limited to PEG-c-DOMG, PEG-DMG. Thefusogenic lipid may be DSPC. See also, Schrum et al., Delivery andFormulation of Engineered Nucleic Acids, US published application20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

US Patent Publication No. 20050019923 describes cationic dendrimers fordelivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step-wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesised from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropylenimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropylenimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine. andphosphorous containing compounds with a mixture of amine/amide orN-P(O2)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropylenimine dendrimersfor gene delivery. Polypropylenimine dendrimers have also been studiedas pH sensitive controlled release systems for drug delivery and fortheir encapsulation of guest molecules when chemically modified byperipheral amino acid groups. The cytotoxicity and interaction ofpolypropylenimine dendrimers with DNA as well as the transfectionefficacy of DAB 64 has also been studied.

US Patent Publication No. 20050019923 is based upon the observationthat, contrary to earlier reports, cationic dendrimers, such aspolypropylenimine dendrimers, display suitable properties, such asspecific targeting and low toxicity, for use in the targeted delivery ofbioactive molecules, such as genetic material. In addition, derivativesof the cationic dendrimer also display suitable properties for thetargeted delivery of bioactive molecules. See also, Bioactive Polymers,US published application 20080267903, which discloses “Various polymers,including cationic polyamine polymers and dendrimeric polymers, areshown to possess anti-proliferative activity, and may therefore beuseful for treatment of disorders characterised by undesirable cellularproliferation such as neoplasms and tumours, inflammatory disorders(including autoimmune disorders), psoriasis and atherosclerosis. Thepolymers may be used alone as active agents, or as delivery vehicles forother therapeutic agents, such as drug molecules or nucleic acids forgene therapy. In such cases, the polymers' own intrinsic anti-tumouractivity may complement the activity of the agent to be delivered.” Thedisclosures of these patent publications may be employed in conjunctionwith herein teachings for delivery of CRISPR Cas system(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of CRISPR Cas system(s) or component(s)thereof or nucleic acid molecule(s) coding therefor. Bothsupernegatively and superpositively charged proteins exhibit aremarkable ability to withstand thermally or chemically inducedaggregation. Superpositively charged proteins are also able to penetratemammalian cells. Associating cargo with these proteins, such as plasmidDNA, RNA, or other proteins, can enable the functional delivery of thesemacromolecules into mammalian cells both in vitro and in vivo. DavidLiu's lab reported the creation and characterization of superchargedproteins in 2007 (Lawrence et al., 2007, Journal of the AmericanChemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or othersuperpositively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116) (However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines):

-   -   (1) One day before treatment, plate 1×105 cells per well in a        48-well plate.    -   (2) On the day of treatment, dilute purified +36 GFP protein in        serumfree media to a final concentration 200 nM. Add RNA to a        final concentration of 50 nM. Vortex to mix and incubate at room        temperature for 10 min.    -   (3) During incubation, aspirate media from cells and wash once        with PBS.    -   (4) Following incubation of +36 GFP and RNA, add the protein-RNA        complexes to cells.    -   (5) Incubate cells with complexes at 37° C. for 4h.    -   (6) Following incubation, aspirate the media and wash three        times with 20 U/mL heparin PBS. Incubate cells with        serum-containing media for a further 48h or longer depending        upon the assay for activity.    -   (7) Analyze cells by immunoblot, qPCR, phenotypic assay, or        other appropriate method.

David Liu's lab has further found +36 GFP to be an effective plasmiddelivery reagent in a range of cells. As plasmid DNA is a larger cargothan siRNA, proportionately more +36 GFP protein is required toeffectively complex plasmids. For effective plasmid delivery Applicantshave developed a variant of +36 GFP bearing a C-terminal HA2 peptidetag, a known endosome-disrupting peptide derived from the influenzavirus hemagglutinin protein. The following protocol has been effectivein a variety of cells, but as above it is advised that plasmid DNA andsupercharged protein doses be optimized for specific cell lines anddelivery applications:

-   -   (1) One day before treatment, plate 1×105 per well in a 48-well        plate.    -   (2) On the day of treatment, dilute purified b36 GFP protein in        serumfree media to a final concentration 2 mM. Add 1 mg of        plasmid DNA. Vortex to mix and incubate at room temperature for        10 min.    -   (3) During incubation, aspirate media from cells and wash once        with PBS.    -   (4) Following incubation of 36 GFP and plasmid DNA, gently add        the protein-DNA complexes to cells.    -   (5) Incubate cells with complexes at 37 C for 4h.    -   (6) Following incubation, aspirate the media and wash with PBS.        Incubate cells in serum-containing media and incubate for a        further 24-48h.    -   (7) Analyze plasmid delivery (e.g., by plasmid-driven gene        expression) as appropriate. See also, e.g., McNaughton et al.,        Proc. Natl. Acad. Sci. USA 106, 6111-6116 (2009); Cronican et        al., ACS Chemical Biology 5, 747-752 (2010); Cronican et al.,        Chemistry & Biology 18, 833-838 (2011); Thompson et al., Methods        in Enzymology 503, 293-319 (2012); Thompson, D. B., et al.,        Chemistry & Biology 19 (7), 831-843 (2012). The methods of the        super charged proteins may be used and/or adapted for delivery        of the CRISPR Cas system of the present invention. These systems        of Dr. Lui and documents herein in conjunction with herein        teaching can be employed in the delivery of CRISPR Cas system(s)        or component(s) thereof or nucleic acid molecule(s) coding        therefor.

Cell Penetrating Peptides (CPPs)

In yet another embodiment, cell penetrating peptides (CPPs) arecontemplated for the delivery of the CRISPR Cas system. CPPs are shortpeptides that facilitate cellular uptake of various molecular cargo(from nanosize particles to small chemical molecules and large fragmentsof DNA). The term “cargo” as used herein includes but is not limited tothe group consisting of therapeutic agents, diagnostic probes, peptides,nucleic acids, antisense oligonucleotides, plasmids, proteins,particles, including nanoparticles, liposomes, chromophores, smallmolecules and radioactive materials. In aspects of the invention, thecargo may also comprise any component of the CRISPR Cas system or theentire functional CRISPR Cas system. Aspects of the present inventionfurther provide methods for delivering a desired cargo into a subjectcomprising: (a) preparing a complex comprising the cell penetratingpeptide of the present invention and a desired cargo, and (b) orally,intraarticularly, intraperitoneally, intrathecally, intrarterially,intranasally, intraparenchymally, subcutaneously, intramuscularly,intravenously, dermally, intrarectally, or topically administering thecomplex to a subject. The cargo is associated with the peptides eitherthrough chemical linkage via covalent bonds or through non-covalentinteractions.

The function of the CPPs are to deliver the cargo into cells, a processthat commonly occurs through endocytosis with the cargo delivered to theendosomes of living mammalian cells. Cell-penetrating peptides are ofdifferent sizes, amino acid sequences, and charges but all CPPs have onedistinct characteristic, which is the ability to translocate the plasmamembrane and facilitate the delivery of various molecular cargoes to thecytoplasm or an organelle. CPP translocation may be classified intothree main entry mechanisms: direct penetration in the membrane,endocytosis-mediated entry, and translocation through the formation of atransitory structure. CPPs have found numerous applications in medicineas drug delivery agents in the treatment of different diseases includingcancer and virus inhibitors, as well as contrast agents for celllabeling. Examples of the latter include acting as a carrier for GFP,MRI contrast agents, or quantum dots. CPPs hold great potential as invitro and in vivo delivery vectors for use in research and medicine.CPPs typically have an amino acid composition that either contains ahigh relative abundance of positively charged amino acids such as lysineor arginine or has sequences that contain an alternating pattern ofpolar/charged amino acids and non-polar, hydrophobic amino acids. Thesetwo types of structures are referred to as polycationic or amphipathic,respectively. A third class of CPPs are the hydrophobic peptides,containing only apolar residues, with low net charge or have hydrophobicamino acid groups that are crucial for cellular uptake. One of theinitial CPPs discovered was the trans-activating transcriptionalactivator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) which wasfound to be efficiently taken up from the surrounding media by numerouscell types in culture. Since then, the number of known CPPs has expandedconsiderably and small molecule synthetic analogues with more effectiveprotein transduction properties have been generated. CPPs include butare not limited to Penetratin, Tat (48-60), Transportan, and (R-AhX-R4)(Ahx=aminohexanoyl).

U.S. Pat. No. 8,372,951, provides a CPP derived from eosinophil cationicprotein (ECP) which exhibits highly cell-penetrating efficiency and lowtoxicity. Aspects of delivering the CPP with its cargo into a vertebratesubject are also provided. Further aspects of CPPs and their deliveryare described in U.S. Pat. Nos. 8,575,305; 8; 614,194 and 8,044,019.CPPs can be used to deliver the CRISPR-Cas system or components thereof.That CPPs can be employed to deliver the CRISPR-Cas system or componentsthereof is also provided in the manuscript “Gene disruption bycell-penetrating peptide-mediated delivery of Cas9 protein and guideRNA”, by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, etal. Genome Res. 2014 Apr. 2. [Epub ahead of print], incorporated byreference in its entirety, wherein it is demonstrated that treatmentwith CPP-conjugated recombinant Cas9 protein and CPP-complexed guideRNAs lead to endogenous gene disruptions in human cell lines. In thepaper the Cas9 protein was conjugated to CPP via a thioether bond,whereas the guide RNA was complexed with CPP, forming condensed,positively charged particles. It was shown that simultaneous andsequential treatment of human cells, including embryonic stem cells,dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinomacells, with the modified Cas9 and guide RNA led to efficient genedisruptions with reduced off-target mutations relative to plasmidtransfections.

Implantable Devices

In another embodiment, implantable devices are also contemplated fordelivery of the CRISPR Cas system or component(s) thereof or nucleicacid molecule(s) coding therefor. For example, US Patent Publication20110195123 discloses an implantable medical device which elutes a druglocally and in prolonged period is provided, including several types ofsuch a device, the treatment modes of implementation and methods ofimplantation. The device comprising of polymeric substrate, such as amatrix for example, that is used as the device body, and drugs, and insome cases additional scaffolding materials, such as metals oradditional polymers, and materials to enhance visibility and imaging. Animplantable delivery device can be advantageous in providing releaselocally and over a prolonged period, where drug is released directly tothe extracellular matrix (ECM) of the diseased area such as tumor,inflammation, degeneration or for symptomatic objectives, or to injuredsmooth muscle cells, or for prevention. One kind of drug is RNA, asdisclosed above, and this system may be used/and or adapted to theCRISPR Cas system of the present invention. The modes of implantation insome embodiments are existing implantation procedures that are developedand used today for other treatments, including brachytherapy and needlebiopsy. In such cases the dimensions of the new implant described inthis invention are similar to the original implant. Typically a fewdevices are implanted during the same treatment procedure.

US Patent Publication 20110195123, provides a drug delivery implantableor insertable system, including systems applicable to a cavity such asthe abdominal cavity and/or any other type of administration in whichthe drug delivery system is not anchored or attached, comprising abiostable and/or degradable and/or bioabsorbable polymeric substrate,which may for example optionally be a matrix. It should be noted thatthe term “insertion” also includes implantation. The drug deliverysystem is preferably implemented as a “Loder” as described in US PatentPublication 20110195123.

The polymer or plurality of polymers are biocompatible, incorporating anagent and/or plurality of agents, enabling the release of agent at acontrolled rate, wherein the total volume of the polymeric substrate,such as a matrix for example, in some embodiments is optionally andpreferably no greater than a maximum volume that permits a therapeuticlevel of the agent to be reached. As a non-limiting example, such avolume is preferably within the range of 0.1 m3 to 1000 mm3, as requiredby the volume for the agent load. The Loder may optionally be larger,for example when incorporated with a device whose size is determined byfunctionality, for example and without limitation, a knee joint, anintra-uterine or cervical ring and the like.

The drug delivery system (for delivering the composition) is designed insome embodiments to preferably employ degradable polymers, wherein themain release mechanism is bulk erosion; or in some embodiments, nondegradable, or slowly degraded polymers are used, wherein the mainrelease mechanism is diffusion rather than bulk erosion, so that theouter part functions as membrane, and its internal part functions as adrug reservoir, which practically is not affected by the surroundingsfor an extended period (for example from about a week to about a fewmonths). Combinations of different polymers with different releasemechanisms may also optionally be used. The concentration gradient atthe surface is preferably maintained effectively constant during asignificant period of the total drug releasing period, and therefore thediffusion rate is effectively constant (termed “zero mode” diffusion).By the term “constant” it is meant a diffusion rate that is preferablymaintained above the lower threshold of therapeutic effectiveness, butwhich may still optionally feature an initial burst and/or mayfluctuate, for example increasing and decreasing to a certain degree.The diffusion rate is preferably so maintained for a prolonged period,and it can be considered constant to a certain level to optimize thetherapeutically effective period, for example the effective silencingperiod.

The drug delivery system optionally and preferably is designed to shieldthe nucleotide based therapeutic agent from degradation, whetherchemical in nature or due to attack from enzymes and other factors inthe body of the subject.

The drug delivery system of US Patent Publication 20110195123 isoptionally associated with sensing and/or activation appliances that areoperated at and/or after implantation of the device, by non and/orminimally invasive methods of activation and/oracceleration/deceleration, for example optionally including but notlimited to thermal heating and cooling, laser beams, and ultrasonic,including focused ultrasound and/or RF (radiofrequency) methods ordevices.

According to some embodiments of US Patent Publication 20110195123, thesite for local delivery may optionally include target sitescharacterized by high abnormal proliferation of cells, and suppressedapoptosis, including tumors, active and or chronic inflammation andinfection including autoimmune diseases states, degenerating tissueincluding muscle and nervous tissue, chronic pain, degenerative sites,and location of bone fractures and other wound locations for enhancementof regeneration of tissue, and injured cardiac, smooth and striatedmuscle.

The site for implantation of the composition, or target site, preferablyfeatures a radius, area and/or volume that is sufficiently small fortargeted local delivery. For example, the target site optionally has adiameter in a range of from about 0.1 mm to about 5 cm.

The location of the target site is preferably selected for maximumtherapeutic efficacy. For example, the composition of the drug deliverysystem (optionally with a device for implantation as described above) isoptionally and preferably implanted within or in the proximity of atumor environment, or the blood supply associated thereof.

For example the composition (optionally with the device) is optionallyimplanted within or in the proximity to pancreas, prostate, breast,liver, via the nipple, within the vascular system and so forth.

The target location is optionally selected from the group comprising,consisting essentially of, or consisting of (as non-limiting examplesonly, as optionally any site within the body may be suitable forimplanting a Loder): 1. brain at degenerative sites like in Parkinson orAlzheimer disease at the basal ganglia, white and gray matter; 2. spineas in the case of amyotrophic lateral sclerosis (ALS); 3. uterine cervixto prevent HPV infection; 4. active and chronic inflammatory joints; 5.dermis as in the case of psoriasis; 6. sympathetic and sensoric nervoussites for analgesic effect; 7. Intra osseous implantation; 8. acute andchronic infection sites; 9. Intra vaginal; 10. Inner ear-auditorysystem, labyrinth of the inner ear, vestibular system; 11. Intratracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urinary bladder;14. biliary system; 15. parenchymal tissue including and not limited tothe kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18.dental gums; 19. Intra-articular (into joints); 20. Intra-ocular; 21.Brain tissue; 22. Brain ventricles; 23. Cavities, including abdominalcavity (for example but without limitation, for ovary cancer); 24. Intraesophageal and 25. Intra rectal.

Optionally insertion of the system (for example a device containing thecomposition) is associated with injection of material to the ECM at thetarget site and the vicinity of that site to affect local pH and/ortemperature and/or other biological factors affecting the diffusion ofthe drug and/or drug kinetics in the ECM, of the target site and thevicinity of such a site.

Optionally, according to some embodiments, the release of said agentcould be associated with sensing and/or activation appliances that areoperated prior and/or at and/or after insertion, by non and/or minimallyinvasive and/or else methods of activation and/oracceleration/deceleration, including laser beam, radiation, thermalheating and cooling, and ultrasonic, including focused ultrasound and/orRF (radiofrequency) methods or devices, and chemical activators.

According to other embodiments of US Patent Publication 20110195123, thedrug preferably comprises a RNA, for example for localized cancer casesin breast, pancreas, brain, kidney, bladder, lung, and prostate asdescribed below. Although exemplified with RNAi, many drugs areapplicable to be encapsulated in Loder, and can be used in associationwith this invention, as long as such drugs can be encapsulated with theLoder substrate, such as a matrix for example, and this system may beused and/or adapted to deliver the CRISPR Cas system of the presentinvention.

As another example of a specific application, neuro and musculardegenerative diseases develop due to abnormal gene expression. Localdelivery of RNAs may have therapeutic properties for interfering withsuch abnormal gene expression. Local delivery of anti apoptotic, antiinflammatory and anti degenerative drugs including small drugs andmacromolecules may also optionally be therapeutic. In such cases theLoder is applied for prolonged release at constant rate and/or through adedicated device that is implanted separately. All of this may be usedand/or adapted to the CRISPR Cas system of the present invention.

As yet another example of a specific application, psychiatric andcognitive disorders are treated with gene modifiers. Gene knockdown is atreatment option. Loders locally delivering agents to central nervoussystem sites are therapeutic options for psychiatric and cognitivedisorders including but not limited to psychosis, bi-polar diseases,neurotic disorders and behavioral maladies. The Loders could alsodeliver locally drugs including small drugs and macromolecules uponimplantation at specific brain sites. All of this may be used and/oradapted to the CRISPR Cas system of the present invention.

As another example of a specific application, silencing of innate and/oradaptive immune mediators at local sites enables the prevention of organtransplant rejection. Local delivery of RNAs and immunomodulatingreagents with the Loder implanted into the transplanted organ and/or theimplanted site renders local immune suppression by repelling immunecells such as CD8 activated against the transplanted organ. All of thismay be used/and or adapted to the CRISPR Cas system of the presentinvention.

As another example of a specific application, vascular growth factorsincluding VEGFs and angiogenin and others are essential forneovascularization. Local delivery of the factors, peptides,peptidomimetics, or suppressing their repressors is an importanttherapeutic modality; silencing the repressors and local delivery of thefactors, peptides, macromolecules and small drugs stimulatingangiogenesis with the Loder is therapeutic for peripheral, systemic andcardiac vascular disease.

The method of insertion, such as implantation, may optionally already beused for other types of tissue implantation and/or for insertions and/orfor sampling tissues, optionally without modifications, or alternativelyoptionally only with non-major modifications in such methods. Suchmethods optionally include but are not limited to brachytherapy methods,biopsy, endoscopy with and/or without ultrasound, such as ERCP,stereotactic methods into the brain tissue, Laparoscopy, includingimplantation with a laparoscope into joints, abdominal organs, thebladder wall and body cavities.

Implantable devices may also include cells, such as epidermal progenitorcells that have been edited or modified to express the CRISPR-Cassystems disclosed herein and embedded with an implantable device, suchas a patch. See. Yue et al. “Engineered Epidermal Progenitor Cells CanCorrect Diet-Induced Obesity and Diabetes” Cell Stem Cell (2017)21(2):256-263.

Implantable device technology herein discussed can be employed withherein teachings and hence by this disclosure and the knowledge in theart, CRISPR-Cas system or components thereof or nucleic acid moleculesthereof or encoding or providing components may be delivered via animplantable device.

Aerosol Delivery

Subjects treated for a lung disease may for example receivepharmaceutically effective amount of aerosolized AAV vector system perlung endobronchially delivered while spontaneously breathing. As such,aerosolized delivery is preferred for AAV delivery in general. Anadenovirus or an AAV particle may be used for delivery. Suitable geneconstructs, each operably linked to one or more regulatory sequences,may be cloned into the delivery vector.

Hybrid Viral Capsid Delivery Systems

In one aspect, the invention provides a particle delivery systemcomprising a hybrid virus capsid protein or hybrid viral outer protein,wherein the hybrid virus capsid or outer protein comprises a viruscapsid or outer protein attached to at least a portion of a non-capsidprotein or peptide. The genetic material of a virus is stored within aviral structure called the capsid. The capsid of certain viruses areenclosed in a membrane called the viral envelope. The viral envelope ismade up of a lipid bilayer embedded with viral proteins including viralglycoproteins. As used herein, an “envelope protein” or “outer protein”means a protein exposed at the surface of a viral particle that is not acapsid protein. For example envelope or outer proteins typicallycomprise proteins embedded in the envelope of the virus. Non-limitingexamples of outer or envelope proteins include, without limit, gp41 andgp120 of HIV, hemagglutinin, neuraminidase and M2 proteins of influenzavirus.

In one example embodiment of the delivery system, the non-capsid proteinor peptide has a molecular weight of up to a megadalton, or has amolecular weight in the range of 110 to 160 kDa, 160 to 200 kDa, 200 to250 kDa, 250 to 300 kDa, 300 to 400 kDa, or 400 to 500 kDa, thenon-capsid protein or peptide comprises a CRISPR protein.

The present application provides a vector for delivering an effectorprotein and at least one CRISPR guide RNA to a cell comprising a minimalpromoter operably linked to a polynucleotide sequence encoding theeffector protein and a second minimal promoter operably linked to apolynucleotide sequence encoding at least one guide RNA, wherein thelength of the vector sequence comprising the minimal promoters andpolynucleotide sequences is less than 4.4Kb. In an embodiment, the virusis an adeno-associated virus (AAV) or an adenovirus. In anotherembodiment, the effector protein is a CRISPR anzyme. In a furtherembodiment, the CRISPR enzyme is SaCas9, Cpf1, Cas13b or C2c2.

In a related aspect, the invention provides a lentiviral vector fordelivering an effector protein and at least one CRISPR guide RNA to acell comprising a promoter operably linked to a polynucleotide sequenceencoding Cpf1 and a second promoter operably linked to a polynucleotidesequence encoding at least one guide RNA, wherein the polynucleotidesequences are in reverse orientation.

In an embodiment of the delivery system, the virus is lentivirus ormurine leukemia virus (MuMLV).

In an embodiment of the delivery system, the virus is an Adenoviridae ora Parvoviridae or a retrovirus or a Rhabdoviridae or an enveloped virushaving a glycoprotein protein (G protein).

In an embodiment of the delivery system, the virus is VSV or rabiesvirus.

In an embodiment of the delivery system, the capsid or outer proteincomprises a capsid protein having VP1, VP2 or VP3.

In an embodiment of the delivery system, the capsid protein is VP3, andthe non-capsid protein is inserted into or attached to VP3 loop 3 orloop 6.

In an embodiment of the delivery system, the virus is delivered to theinterior of a cell.

In an embodiment of the delivery system, the capsid or outer protein andthe non-capsid protein can dissociate after delivery into a cell.

In an embodiment of the delivery system, the capsid or outer protein isattached to the protein by a linker.

In an embodiment of the delivery system, the linker comprises aminoacids.

In an embodiment of the delivery system, the linker is a chemicallinker.

In an embodiment of the delivery system, the linker is cleavable.

In an embodiment of the delivery system, the linker is biodegradable.

In an embodiment of the delivery system, the linker comprises(GGGGS)1-3, ENLYFQG, or a disulfide.

In an embodiment, the delivery system comprises a protease or nucleicacid molecule(s) encoding a protease that is expressed, said proteasebeing capable of cleaving the linker, whereby there can be cleavage ofthe linker. In an embodiment of the invention, a protease is deliveredwith a particle component of the system, for example packaged, mixedwith, or enclosed by lipid and or capsid. Entry of the particle into acell is thereby accompanied or followed by cleavage and dissociation ofpayload from particle. In certain embodiments, an expressible nucleicacid encoding a protease is delivered, whereby at entry or followingentry of the particle into a cell, there is protease expression, linkercleavage, and dissociation of payload from capsid. In certainembodiments, dissociation of payload occurs with viral replication. Incertain embodiments, dissociation of payload occurs in the absence ofproductive virus replication.

In an embodiment of the delivery system, each terminus of a CRISPRprotein is attached to the capsid or outer protein by a linker.

In an embodiment of the delivery system, the non-capsid protein isattached to the exterior portion of the capsid or outer protein.

In an embodiment of the delivery system, the non-capsid protein isattached to the interior portion of the capsid or outer protein.

In an embodiment of the delivery system, the capsid or outer protein andthe non-capsid protein are a fusion protein.

In an embodiment of the delivery system, the non-capsid protein isencapsulated by the capsid or outer protein.

In an embodiment of the delivery system, the non-capsid protein isattached to a component of the capsid protein or a component of theouter protein prior to formation of the capsid or the outer protein.

In an embodiment of the delivery system, the protein is attached to thecapsid or outer protein after formation of the capsid or outer protein.

In an embodiment, the delivery system comprises a targeting moiety, suchas active targeting of a lipid entity of the invention, e.g., lipidparticle or nanoparticle or liposome or lipid bylayer of the inventioncomprising a targeting moiety for active targeting.

With regard to targeting moieties, mention is made of Deshpande et al,“Current trends in the use of liposomes for tumor targeting,”Nanomedicine (Lond). 8(9), doi:10.2217/nnm.13.118 (2013), and thedocuments it cites, all of which are incorporated herein by reference.Mention is also made of WO/2016/027264, and the documents it cites, allof which are incorporated herein by reference. And mention is made ofLorenzer et al, “Going beyond the liver: Progress and challenges oftargeted delivery of siRNA therapeutics,” Journal of Controlled Release,203: 1-15 (2015), and the documents it cites, all of which areincorporated herein by reference.

An actively targeting lipid particle or nanoparticle or liposome orlipid bylayer delivery system (generally as to embodiments of theinvention, “lipid entity of the invention” delivery systems) areprepared by conjugating targeting moieties, including small moleculeligands, peptides and monoclonal antibodies, on the lipid or liposomalsurface; for example, certain receptors, such as folate and transferrin(Tf) receptors (TfR), are overexpressed on many cancer cells and havebeen used to make liposomes tumor cell specific. Liposomes thataccumulate in the tumor microenvironment can be subsequently endocytosedinto the cells by interacting with specific cell surface receptors. Toefficiently target liposomes to cells, such as cancer cells, it isuseful that the targeting moiety have an affinity for a cell surfacereceptor and to link the targeting moiety in sufficient quantities tohave optimum affinity for the cell surface receptors; and determiningthese aspects are within the ambit of the skilled artisan. In the fieldof active targeting, there are a number of cell-, e.g., tumor-, specifictargeting ligands.

Also as to active targeting, with regard to targeting cell surfacereceptors such as cancer cell surface receptors, targeting ligands onliposomes can provide attachment of liposomes to cells, e.g., vascularcells, via a noninternalizing epitope; and, this can increase theextracellular concentration of that which is being delivered, therebyincreasing the amount delivered to the target cells. A strategy totarget cell surface receptors, such as cell surface receptors on cancercells, such as overexpressed cell surface receptors on cancer cells, isto use receptor-specific ligands or antibodies. Many cancer cell typesdisplay upregulation of tumor-specific receptors. For example, TfRs andfolate receptors (FRs) are greatly overexpressed by many tumor celltypes in response to their increased metabolic demand. Folic acid can beused as a targeting ligand for specialized delivery owing to its ease ofconjugation to nanocarriers, its high affinity for FRs and therelatively low frequency of FRs, in normal tissues as compared withtheir overexpression in activated macrophages and cancer cells, e.g.,certain ovarian, breast, lung, colon, kidney and brain tumors.Overexpression of FR on macrophages is an indication of inflammatorydiseases, such as psoriasis, Crohn's disease, rheumatoid arthritis andatherosclerosis; accordingly, folate-mediated targeting of the inventioncan also be used for studying, addressing or treating inflammatorydisorders, as well as cancers. Folate-linked lipid particles ornanoparticles or liposomes or lipid by layers of the invention (“lipidentity of the invention”) deliver their cargo intracellularly throughreceptor-mediated endocytosis. Intracellular trafficking can be directedto acidic compartments that facilitate cargo release, and, mostimportantly, release of the cargo can be altered or delayed until itreaches the cytoplasm or vicinity of target organelles. Delivery ofcargo using a lipid entity of the invention having a targeting moiety,such as a folate-linked lipid entity of the invention, can be superiorto nontargeted lipid entity of the invention. The attachment of folatedirectly to the lipid head groups may not be favorable for intracellulardelivery of folate-conjugated lipid entity of the invention, since theymay not bind as efficiently to cells as folate attached to the lipidentity of the invention surface by a spacer, which may can enter cancercells more efficiently. A lipid entity of the invention coupled tofolate can be used for the delivery of complexes of lipid, e.g.,liposome, e.g., anionic liposome and virus or capsid or envelope orvirus outer protein, such as those herein discussed such as adenovirousor AAV. Tf is a monomeric serum glycoprotein of approximately 80 KDainvolved in the transport of iron throughout the body. Tf binds to theTfR and translocates into cells via receptor-mediated endocytosis. Theexpression of TfR is can be higher in certain cells, such as tumor cells(as compared with normal cells and is associated with the increased irondemand in rapidly proliferating cancer cells. Accordingly, the inventioncomprehends a TfR-targeted lipid entity of the invention, e.g., as toliver cells, liver cancer, breast cells such as breast cancer cells,colon such as colon cancer cells, ovarian cells such as ovarian cancercells, head, neck and lung cells, such as head, neck and non-small-celllung cancer cells, cells of the mouth such as oral tumor cells.

Also as to active targeting, a lipid entity of the invention can bemultifunctional, i.e., employ more than one targeting moiety such asCPP, along with Tf; a bifunctional system; e.g., a combination of Tf andpoly-L-arginine which can provide transport across the endothelium ofthe blood-brain barrier. EGFR, is a tyrosine kinase receptor belongingto the ErbB family of receptors that mediates cell growth,differentiation and repair in cells, especially non-cancerous cells, butEGF is overexpressed in certain cells such as many solid tumors,including colorectal, non-small-cell lung cancer, squamous cellcarcinoma of the ovary, kidney, head, pancreas, neck and prostate, andespecially breast cancer. The invention comprehends EGFR-targetedmonoclonal antibody(ies) linked to a lipid entity of the invention.HER-2 is often overexpressed in patients with breast cancer, and is alsoassociated with lung, bladder, prostate, brain and stomach cancers.HER-2, encoded by the ERBB2 gene. The invention comprehends aHER-2-targeting lipid entity of the invention, e.g., ananti-HER-2-antibody(or binding fragment thereof)-lipid entity of theinvention, a HER-2-targeting-PEGylated lipid entity of the invention(e.g., having an anti-HER-2-antibody or binding fragment thereof), aHER-2-targeting-maleimide-PEG polymer- lipid entity of the invention(e.g., having an anti-HER-2-antibody or binding fragment thereof). Uponcellular association, the receptor-antibody complex can be internalizedby formation of an endosome for delivery to the cytoplasm. With respectto receptor-mediated targeting, the skilled artisan takes intoconsideration ligand/target affinity and the quantity of receptors onthe cell surface, and that PEGylation can act as a barrier againstinteraction with receptors. The use of antibody-lipid entity of theinvention targeting can be advantageous. Multivalent presentation oftargeting moieties can also increase the uptake and signaling propertiesof antibody fragments. In practice of the invention, the skilled persontakes into account ligand density (e.g., high ligand densities on alipid entity of the invention may be advantageous for increased bindingto target cells). Preventing early by macrophages can be addressed witha sterically stabilized lipid entity of the invention and linkingligands to the terminus of molecules such as PEG, which is anchored inthe lipid entity of the invention (e.g., lipid particle or nanoparticleor liposome or lipid bylayer). The microenvironment of a cell mass suchas a tumor microenvironment can be targeted; for instance, it may beadvantageous to target cell mass vasculature, such as the tumorvasculature microenvironment. Thus, the invention comprehends targetingVEGF. VEGF and its receptors are well-known proangiogenic molecules andare well-characterized targets for antiangiogenic therapy. Manysmall-molecule inhibitors of receptor tyrosine kinases, such as VEGFRsor basic FGFRs, have been developed as anticancer agents and theinvention comprehends coupling any one or more of these peptides to alipid entity of the invention, e.g., phage IVO peptide(s) (e.g., via orwith a PEG terminus), tumor-homing peptide APRPG such asAPRPG-PEG-modified. VCAM, the vascular endothelium plays a key role inthe pathogenesis of inflammation, thrombosis and atherosclerosis. CAMsare involved in inflammatory disorders, including cancer, and are alogical target, E- and P-selectins, VCAM-1 and ICAMs. Can be used totarget a lipid entity of the invention., e.g., with PEGylation. Matrixmetalloproteases (MMPs) belong to the family of zinc-dependentendopeptidases. They are involved in tissue remodeling, tumorinvasiveness, resistance to apoptosis and metastasis. There are four MMPinhibitors called TIMP1-4, which determine the balance between tumorgrowth inhibition and metastasis; a protein involved in the angiogenesisof tumor vessels is MT1-MMP, expressed on newly formed vessels and tumortissues. The proteolytic activity of MT1-MMP cleaves proteins, such asfibronectin, elastin, collagen and laminin, at the plasma membrane andactivates soluble MMPs, such as MMP-2, which degrades the matrix. Anantibody or fragment thereof such as a Fab′ fragment can be used in thepractice of the invention such as for an antihuman MT1-MMP monoclonalantibody linked to a lipid entity of the invention, e.g., via a spacersuch as a PEG spacer. αβ-integrins or integrins are a group oftransmembrane glycoprotein receptors that mediate attachment between acell and its surrounding tissues or extracellular matrix. Integrinscontain two distinct chains (heterodimers) called α- and β-subunits. Thetumor tissue-specific expression of integrin receptors can be utilizedfor targeted delivery in the invention, e.g., whereby the targetingmoiety can be an RGD peptide such as a cyclic RGD. Aptamers are ssDNA orRNA oligonucleotides that impart high affinity and specific recognitionof the target molecules by electrostatic interactions, hydrogen bondingand hydro phobic interactions as opposed to the Watson-Crick basepairing, which is typical for the bonding interactions ofoligonucleotides. Aptamers as a targeting moiety can have advantagesover antibodies: aptamers can demonstrate higher target antigenrecognition as compared with antibodies; aptamers can be more stable andsmaller in size as compared with antibodies; aptamers can be easilysynthesized and chemically modified for molecular conjugation; andaptamers can be changed in sequence for improved selectivity and can bedeveloped to recognize poorly immunogenic targets. Such moieties as asgc8 aptamer can be used as a targeting moiety (e.g., via covalentlinking to the lipid entity of the invention, e.g., via a spacer, suchas a PEG spacer). The targeting moiety can be stimuli-sensitive, e.g.,sensitive to an externally applied stimuli, such as magnetic fields,ultrasound or light; and pH-triggering can also be used, e.g., a labilelinkage can be used between a hydrophilic moiety such as PEG and ahydrophobic moiety such as a lipid entity of the invention, which iscleaved only upon exposure to the relatively acidic conditionscharacteristic of the a particular environment or microenvironment suchas an endocytic vacuole or the acidotic tumor mass. pH-sensitivecopolymers can also be incorporated in embodiments of the invention canprovide shielding; diortho esters, vinyl esters, cysteine-cleavablelipopolymers, double esters and hydrazones are a few examples ofpH-sensitive bonds that are quite stable at pH 7.5, but are hydrolyzedrelatively rapidly at pH 6 and below, e.g., a terminally alkylatedcopolymer of N-isopropylacrylamide and methacrylic acid that copolymerfacilitates destabilization of a lipid entity of the invention andrelease in compartments with decreased pH value; or, the inventioncomprehends ionic polymers for generation of a pH-responsive lipidentity of the invention (e.g., poly(methacrylic acid),poly(diethylaminoethyl methacrylate), poly(acrylamide) and poly(acrylicacid)). Temperature-triggered delivery is also within the ambit of theinvention. Many pathological areas, such as inflamed tissues and tumors,show a distinctive hyperthermia compared with normal tissues. Utilizingthis hyperthermia is an attractive strategy in cancer therapy sincehyperthermia is associated with increased tumor permeability andenhanced uptake. This technique involves local heating of the site toincrease microvascular pore size and blood flow, which, in turn, canresult in an increased extravasation of embodiments of the invention.Temperature-sensitive lipid entity of the invention can be prepared fromthermosensitive lipids or polymers with a low critical solutiontemperature. Above the low critical solution temperature (e.g., at sitesuch as tumor site or inflamed tissue site), the polymer precipitates,disrupting the liposomes to release. Lipids with a specificgel-to-liquid phase transition temperature are used to prepare theselipid entities of the invention; and a lipid for a thermosensitiveembodiment can be dipalmitoylphosphatidylcholine. Thermosensitivepolymers can also facilitate destabilization followed by release, and auseful thermosensitive polymer is poly (N-isopropylacrylamide). Anothertemperature triggered system can employ lysolipid temperature-sensitiveliposomes. The invention also comprehends redox-triggered delivery: Thedifference in redox potential between normal and inflamed or tumortissues, and between the intra- and extra-cellular environments has beenexploited for delivery; e.g., GSH is a reducing agent abundant in cells,especially in the cytosol, mitochondria and nucleus. The GSHconcentrations in blood and extracellular matrix are just one out of 100to one out of 1000 of the intracellular concentration, respectively.This high redox potential difference caused by GSH, cysteine and otherreducing agents can break the reducible bonds, destabilize a lipidentity of the invention and result in release of payload. The disulfidebond can be used as the cleavable/reversible linker in a lipid entity ofthe invention, because it causes sensitivity to redox owing to thedisulfideto-thiol reduction reaction; a lipid entity of the inventioncan be made reduction sensitive by using two (e.g., two forms of adisulfide-conjugated multifunctional lipid as cleavage of the disulfidebond (e.g., via tris(2-carboxyethyl)phosphine, dithiothreitol,L-cysteine or GSH), can cause removal of the hydrophilic head group ofthe conjugate and alter the membrane organization leading to release ofpayload. Calcein release from reduction-sensitive lipid entity of theinvention containing a disulfide conjugate can be more useful than areduction-insensitive embodiment. Enzymes can also be used as a triggerto release payload. Enzymes, including MMfPs (e.g. MMP2), phospholipaseA2, alkaline phosphatase, transglutaminase orphosphatidylinositol-specific phospholipase C, have been found to beoverexpressed in certain tissues, e.g., tumor tissues. In the presenceof these enzymes, specially engineered enzyme-sensitive lipid entity ofthe invention can be disrupted and release the payload. anMMP2-cleavable octapeptide (Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln) can beincorporated into a linker, and can have antibody targeting, e.g.,antibody 2C5. The invention also comprehends light-or energy-triggereddelivery, e.g., the lipid entity of the invention can belight-sensitive, such that light or energy can facilitate structural andconformational changes, which lead to direct interaction of the lipidentity of the invention with the target cells via membrane fusion,photo-isomerism, photofragmentation or photopolymerization; such amoiety therefor can be benzoporphyrin photosensitizer. Ultrasound can bea form of energy to trigger delivery; a lipid entity of the inventionwith a small quantity of particular gas, including air or perfluoratedhydrocarbon can be triggered to release with ultrasound, e.g.,low-frequency ultrasound (LFUS). Magnetic delivery: A lipid entity ofthe invention can be magnetized by incorporation of magnetites, such asFe₃O₄ or γ-Fe₂O₃, e.g., those that are less than 10 nm in size. Targeteddelivery can be then by exposure to a magnetic field.

Also as to active targeting, the invention also comprehendsintracellular delivery. Since liposomes follow the endocytic pathway,they are entrapped in the endosomes (pH 6.5-6) and subsequently fusewith lysosomes (pH<5), where they undergo degradation that results in alower therapeutic potential. The low endosomal pH can be taken advantageof to escape degradation. Fusogenic lipids or peptides, whichdestabilize the endosomal membrane after the conformationaltransition/activation at a lowered pH. Amines are protonated at anacidic pH and cause endosomal swelling and rupture by a buffer effectUnsaturated dioleoylphosphatidylethanolamine (DOPE) readily adopts aninverted hexagonal shape at a low pH, which causes fusion of liposomesto the endosomal membrane. This process destabilizes a lipid entitycontaining DOPE and releases the cargo into the cytoplasm; fusogeniclipid GALA, cholesteryl-GALA and PEG-GALA may show a highly efficientendosomal release; a pore-forming protein listeriolysin O may provide anendosomal escape mechanism; and, histidine-rich peptides have theability to fuse with the endosomal membrane, resulting in poreformation, and can buffer the proton pump causing membrane lysis.

Also as to active targeting, cell-penetrating peptides (CPPs) facilitateuptake of macromolecules through cellular membranes and, thus, enhancethe delivery of CPP-modified molecules inside the cell. CPPs can besplit into two classes: amphipathic helical peptides, such astransportan and MAP, where lysine residues are major contributors to thepositive charge; and Arg-rich peptides, such as TATp, Antennapedia orpenetratin. TATp is a transcription-activating factor with 86 aminoacids that contains a highly basic (two Lys and six Arg among nineresidues) protein transduction domain, which brings about nuclearlocalization and RNA binding. Other CPPs that have been used for themodification of liposomes include the following: the minimal proteintransduction domain of Antennapedia, a Drosophilia homeoprotein, calledpenetratin, which is a 16-mer peptide (residues 43-58) present in thethird helix of the homeodomain; a 27-amino acid-long chimeric CPP,containing the peptide sequence from the amino terminus of theneuropeptide galanin bound via the Lys residue, mastoparan, a wasp venompeptide; VP22, a major structural component of HSV-1 facilitatingintracellular transport and transportan (18-mer) amphipathic modelpeptide that translocates plasma membranes of mast cells and endothelialcells by both energy-dependent and -independent mechanisms. Theinvention comprehends a lipid entity of the invention modified withCPP(s), for intracellular delivery that may proceed via energy dependentmacropinocytosis followed by endosomal escape. The invention furthercomprehends organelle-specific targeting. A lipid entity of theinvention surface-functionalized with the triphenylphosphonium (TPP)moiety or a lipid entity of the invention with a lipophilic cation,rhodamine 123 can be effective in delivery of cargo to mitochondria.DOPE/sphingomyelin/stearyl-octa-arginine can delivers cargos to themitochondrial interior via membrane fusion. A lipid entity of theinvention surface modified with a lysosomotropic ligand, octadecylrhodamine B can deliver cargo to lysosomes. Ceramides are useful ininducing lysosomal membrane permeabilization; the invention comprehendsintracellular delivery of a lipid entity of the invention having aceramide. The invention further comprehends a lipid entity of theinvention targeting the nucleus, e.g., via a DNA-intercalating moiety.The invention also comprehends multifunctional liposomes for targeting,i.e., attaching more than one functional group to the surface of thelipid entity of the invention, for instance to enhances accumulation ina desired site and/or promotes organelle-specific delivery and/or targeta particular type of cell and/or respond to the local stimuli such astemperature (e.g., elevated), pH (e.g., decreased), respond toexternally applied stimuli such as a magnetic field, light, energy, heator ultrasound and/or promote intracellular delivery of the cargo. All ofthese are considered actively targeting moieties.

An embodiment of the invention includes the delivery system comprisingan actively targeting lipid particle or nanoparticle or liposome orlipid bilayer delivery system; or comprising a lipid particle ornanoparticle or liposome or lipid bylayer comprising a targeting moietywhereby there is active targeting or wherein the targeting moiety is anactively targeting moiety. A targeting moiety can be one or moretargeting moieties, and a targeting moiety can be for any desired typeof targeting such as, e.g., to target a cell such as anyherein-mentioned; or to target an organelle such as anyherein-mentioned; or for targeting a response such as to a physicalcondition such as heat, energy, ultrasound, light, pH, chemical such asenzymatic, or magnetic stimuli; or to target to achieve a particularoutcome such as delivery of payload to a particular location, such as bycell penetration.

It should be understood that as to each possible targeting or activetargeting moiety herein-discussed, there is an aspect of the inventionwherein the delivery system comprises such a targeting or activetargeting moiety. Likewise, Table 2 provides exemplary targetingmoieties that can be used in the practice of the invention an as to eachan aspect of the invention provides a delivery system that comprisessuch a targeting moiety.

TABLE 2 Targeting Moiety Target Molecule Target Cell or Tissue folatefolate receptor cancer cells transferrin transferrin receptor cancercells Antibody CC52 rat CC531 rat colon adenocarcinoma CC531 anti- HER2antibody HER2 HER2 -overexpressing tumors anti-GD2 GD2 neuroblastoma,melanoma anti-EGFR EGFR tumor cells overexpressing EGFR pH-dependentfusogenic ovarian carcinoma peptide diINF-7 anti-VEGFR VEGF Receptortumor vasculature anti-CD19 CD19 (B cell marker) leukemia, lymphomacell-penetrating peptide blood-brain barrier cyclic arginine-glycine-av33 glioblastoma cells, human umbilical aspartic acid-tyrosine- veinendothelial cells, tumor cysteine peptide angiogenesis (c(RGDyC)-LP)ASSHN peptide endothelial progenitor cells; anti- cancer PR_b peptideα5β1 integrin cancer cells AG86 peptide α6β4 integrin cancer cellsKCCYSL (P6.1 peptide) HER-2 receptor cancer cells affinity peptide LNAminopeptidase N APN-positive tumor (YEVGHRC) (APN/CD13) syntheticsomatostatin Somatostatin receptor 2 breast cancer analogue (SSTR2)anti-CD20 monoclonal B-lymphocytes B cell lymphoma antibody

Thus, in an embodiment of the delivery system, the targeting moietycomprises a receptor ligand, such as, for example, hyaluronic acid forCD44 receptor, galactose for hepatocytes, or antibody or fragmentthereof such as a binding antibody fragment against a desired surfacereceptor, and as to each of a targeting moiety comprising a receptorligand, or an antibody or fragment thereof such as a binding fragmentthereof, such as against a desired surface receptor, there is an aspectof the invention wherein the delivery system comprises a targetingmoiety comprising a receptor ligand, or an antibody or fragment thereofsuch as a binding fragment thereof, such as against a desired surfacereceptor, or hyaluronic acid for CD44 receptor, galactose forhepatocytes (see, e.g., Surace et al, “Lipoplexes targeting the CD44hyaluronic acid receptor for efficient transfection of breast cancercells,” J. Mol Pharm 6(4):1062-73; doi: 10.1021/mp800215d (2009); Sonokeet al, “Galactose-modified cationic liposomes as a liver-targetingdelivery system for small interfering RNA,” Biol Pharm Bull.34(8):1338-42 (2011); Torchilin, “Antibody-modified liposomes for cancerchemotherapy,” Expert Opin. Drug Deliv. 5 (9), 1003-1025 (2008);Manjappa et al, “Antibody derivatization and conjugation strategies:application in preparation of stealth immunoliposome to targetchemotherapeutics to tumor,” J. Control. Release 150 (1), 2-22 (2011);Sofou S “Antibody-targeted liposomes in cancer therapy and imaging,”Expert Opin. Drug Deliv. 5 (2): 189-204 (2008); Gao J et al,“Antibody-targeted immunoliposomes for cancer treatment,” Mini. Rev.Med. Chem. 13(14): 2026-2035 (2013); Molavi et al, “Anti-CD30 antibodyconjugated liposomal doxorubicin with significantly improved therapeuticefficacy against anaplastic large cell lymphoma,” Biomaterials34(34):8718-25 (2013), each of which and the documents cited therein arehereby incorporated herein by reference).

Moreover, in view of the teachings herein the skilled artisan canreadily select and apply a desired targeting moiety in the practice ofthe invention as to a lipid entity of the invention. The inventioncomprehends an embodiment wherein the delivery system comprises a lipidentity having a targeting moiety.

In an embodiment of the delivery system, the protein comprises a CRISPRprotein, or portion thereof.

In some embodiments a non-capsid protein or protein that is not a virusouter protein or a virus envelope (sometimes herein shorthanded as“non-capsid protein”), such as a CRISPR protein or portion thereof, canhave one or more functional moiety(ies) thereon, such as a moiety fortargeting or locating, such as an NLS or NES, or an activator orrepressor.

In an embodiment of the delivery system, a protein or portion thereofcan comprise a tag.

In an aspect, the invention provides a virus particle comprising acapsid or outer protein having one or more hybrid virus capsid or outerproteins comprising the virus capsid or outer protein attached to atleast a portion of a non-capsid protein or a CRISPR protein.

In an aspect, the invention provides an in vitro method of deliverycomprising contacting the delivery system with a cell, optionally aeukaryotic cell, whereby there is delivery into the cell of constituentsof the delivery system.

In an aspect, the invention provides an in vitro, a research or studymethod of delivery comprising contacting the delivery system with acell, optionally a eukaryotic cell, whereby there is delivery into thecell of constituents of the delivery system, obtaining data or resultsfrom the contacting, and transmitting the data or results.

In an aspect, the invention provides a cell from or of an in vitromethod of delivery, wherein the method comprises contacting the deliverysystem with a cell, optionally a eukaryotic cell, whereby there isdelivery into the cell of constituents of the delivery system, andoptionally obtaining data or results from the contacting, andtransmitting the data or results.

In an aspect, the invention provides a cell from or of an in vitromethod of delivery, wherein the method comprises contacting the deliverysystem with a cell, optionally a eukaryotic cell, whereby there isdelivery into the cell of constituents of the delivery system, andoptionally obtaining data or results from the contacting, andtransmitting the data or results; and wherein the cell product isaltered compared to the cell not contacted with the delivery system, forexample altered from that which would have been wild type of the cellbut for the contacting.

In an embodiment, the cell product is non-human or animal.

In one aspect, the invention provides a particle delivery systemcomprising a composite virus particle, wherein the composite virusparticle comprises a lipid, a virus capsid protein, and at least aportion of a non-capsid protein or peptide. The non-capsid peptide orprotein can have a molecular weight of up to one megadalton.

In one embodiment, the particle delivery system comprises a virusparticle adsorbed to a liposome or lipid particle or nanoparticle. Inone embodiment, a virus is adsorbed to a liposome or lipid particle ornanoparticle either through electrostatic interactions, or is covalentlylinked through a linker. The lipid particle or nanoparticles (1 mg/ml)dissolved in either sodium acetate buffer (pH 5.2) or pure H2O (pH 7)are positively charged. The isoelectropoint of most viruses is in therange of 3.5-7. They have a negatively charged surface in either sodiumacetate buffer (pH 5.2) or pure H2O. The electrostatic interactionbetween the virus and the liposome or synthetic lipid nanoparticle isthe most significant factor driving adsorption. By modifying the chargedensity of the lipid nanoparticle, e.g. inclusion of neutral lipids intothe lipid nanoparticle, it is possible to modulate the interactionbetween the lipid nanoparticle and the virus, hence modulating theassembly. In one embodiment, the liposome comprises a cationic lipid.

In one embodiment, the liposome of the particle delivery systemcomprises a CRISPR system component.

In one aspect, the invention provides a delivery system comprising oneor more hybrid virus capsid proteins in combination with a lipidparticle, wherein the hybrid virus capsid protein comprises at least aportion of a virus capsid protein attached to at least a portion of anon-capsid protein.

In one embodiment, the virus capsid protein of the delivery system isattached to a surface of the lipid particle. When the lipid particle isa bilayer, e.g., a liposome, the lipid particle comprises an exteriorhydrophilic surface and an interior hydrophilic surface. In oneembodiment, the virus capsid protein is attached to a surface of thelipid particle by an electrostatic interaction or by hydrophobicinteraction.

In one embodiment, the particle delivery system has a diameter of50-1000 nm, preferably 100-1000 nm.

In one embodiment, the delivery system comprises a non-capsid protein orpeptide, wherein the non-capsid protein or peptide has a molecularweight of up to a megadalton. In one embodiment, the non-capsid proteinor peptide has a molecular weight in the range of 110 to 160 kDa, 160 to200 kDa, 200 to 250 kDa, 250 to 300 kDa, 300 to 400 kDa, or 400 to 500kDa.

In one embodiment, the delivery system comprises a non-capsid protein orpeptide, wherein the protein or peptide comprises a CRISPR protein orpeptide. In one embodiment, the protein or peptide comprises a Cas9, aCpf1 or a C2c2/Cas13a.

In one embodiment, a weight ratio of hybrid capsid protein to wild-typecapsid protein is from 1:10 to 1:1, for example, 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9 and 1:10.

In one embodiment, the virus of the delivery system is an Adenoviridaeor a Parvoviridae or a Rhabdoviridae or an enveloped virus having aglycoprotein protein. In one embodiment, the virus is anadeno-associated virus (AAV) or an adenovirus or a VSV or a rabiesvirus. In one embodiment, the virus is a retrovirus or a lentivirus. Inone embodiment, the virus is murine leukemia virus (MuMLV).

In one embodiment, the virus capsid protein of the delivery systemcomprises VP1, VP2 or VP3.

In one embodiment, the virus capsid protein of the delivery system isVP3, and the non-capsid protein is inserted into or tethered orconnected to VP3 loop 3 or loop 6.

In one embodiment, the virus of the delivery system is delivered to theinterior of a cell.

In one embodiment, the virus capsid protein and the non-capsid proteinare capable of dissociating after delivery into a cell.

In one aspect of the delivery system, the virus capsid protein isattached to the non-capsid protein by a linker. In one embodiment, thelinker comprises amino acids. In one embodiment, the linker is achemical linker. In another embodiment, the linker is cleavable orbiodegradable. In one embodiment, the linker comprises (GGGGS)1-3 (SEQ.I.D. Nos. 5 and 6), ENLYFQG (SEQ. I.D. No. 7), or a disulfide.

In one embodiment of the delivery system, each terminus of thenon-capsid protein is attached to the capsid protein by a linker moiety.

In one embodiment, the non-capsid protein is attached to the exteriorportion of the virus capsid protein. As used herein, “exterior portion”as it refers to a virus capsid protein means the outer surface of thevirus capsid protein when it is in a formed virus capsid.

In one embodiment, the non-capsid protein is attached to the interiorportion of the capsid protein or is encapsulated within the lipidparticle. As used herein, “interior portion” as it refers to a viruscapsid protein means the inner surface of the virus capsid protein whenit is in a formed virus capsid. In one embodiment, the virus capsidprotein and the non-capsid protein are a fusion protein.

In one embodiment, the fusion protein is attached to the surface of thelipid particle.

In one embodiment, the non-capsid protein is attached to the viruscapsid protein prior to formation of the capsid.

In one embodiment, the non-capsid protein is attached to the viruscapsid protein after formation of the capsid.

In one embodiment, the non-capsid protein comprises a targeting moiety.

In one embodiment, the targeting moiety comprises a receptor ligand.

In an embodiment, the non-capsid protein comprises a tag.

In an embodiment, the non-capsid protein comprises one or moreheterologous nuclear localization signals(s) (NLSs).

In an embodiment, the protein or peptide comprises a Type II CRISPRprotein or a Type V CRISPR protein.

In an embodiment, the delivery system further comprises guide RNS,optionally complexed with the CRISPR protein.

In an embodiment, the delivery system comprises a protease or nucleicacid molecule(s) encoding a protease that is expressed, whereby theprotease cleaves the linker. In certain embodiments, there is proteaseexpression, linker cleavage, and dissociation of payload from capsid inthe absence of productive virus replication.

In an aspect, the invention provides a delivery system comprising afirst hybrid virus capsid protein and a second hybrid virus capsidprotein, wherein the first hybrid virus capsid protein comprises a viruscapsid protein attached to a first part of a protein, and wherein thesecond hybrid virus capsid protein comprises a second virus capsidprotein attached to a second part of the protein, wherein the first partof the protein and the second part of the protein are capable ofassociating to form a functional protein.

In an aspect, the invention provides a delivery system comprising afirst hybrid virus capsid protein and a second hybrid virus capsidprotein, wherein the first hybrid virus capsid protein comprises a viruscapsid protein attached to a first part of a CRISPR protein, and whereinthe second hybrid virus capsid protein comprises a second virus capsidprotein attached to a second part of a CRISPR protein, wherein the firstpart of the CRISPR protein and the second part of the CRISPR protein arecapable of associating to form a functional CRISPR protein.

In an embodiment of the delivery system, the first hybrid virus capsidprotein and the second virus capsid protein are on the surface of thesame virus particle.

In an embodiment of the delivery system, the first hybrid virus capsuleprotein is located at the interior of a first virus particle and thesecond hybrid virus capsid protein is located at the interior of asecond virus particle.

In an embodiment of the delivery system, the first part of the proteinor CRISPR protein is linked to a first member of a ligand pair, and thesecond part of the protein or CRISPR protein is linked to a secondmember of a ligand pair, wherein the first part of the ligand pair bindsto the second part of the ligand pair in a cell. In an embodiment, thebinding of the first part of the ligand pair to the second part of theligand pair is inducible.

In an embodiment of the delivery system, either or both of the firstpart of the protein or CRISPR protein and the second part of the proteinor CRISPR protein comprise one or more NLSs.

In an embodiment of the delivery system, either or both of the firstpart of the protein or CRISPR protein and the second part of the proteinor CRISPR protein comprise one or more nuclear export signals (NESs).

In certain embodiments, the virus structural component comprises one ormore capsid proteins including an entire capsid. In certain embodiments,such as wherein a viral capsid comprises multiple copies of differentproteins, the delivery system can provide one or more of the sameprotein or a mixture of such proteins. For example, AAV comprises 3capsid proteins, VP1, VP2, and VP3, thus delivery systems of theinvention can comprise one or more of VP1, and/or one or more of VP2,and/or one or more of VP3. Accordingly, the present invention isapplicable to a virus within the family Adenoviridae, such asAtadenovirus, e.g., Ovine atadenovirus D, Aviadenovirus, e.g., Fowlaviadenovirus A, Ichtadenovirus, e.g., Sturgeon ichtadenovirus A,Mastadenovirus (which includes adenoviruses such as all humanadenoviruses), e.g., Human mastadenovirus C, and Siadenovirus, e.g.,Frog siadenovirus A. Thus, a virus of within the family Adenoviridae iscontemplated as within the invention with discussion herein as toadenovirus applicable to other family members. Target-specific AAVcapsid variants can be used or selected. Non-limiting examples includecapsid variants selected to bind to chronic myelogenous leukemia cells,human CD34 PBPC cells, breast cancer cells, cells of lung, heart, dermalfibroblasts, melanoma cells, stem cell, glioblastoma cells, coronaryartery endothelial cells and keratinocytes. See, e.g., Buning et al,2015, Current Opinion in Pharmacology 24, 94-104. From teachings hereinand knowledge in the art as to modifications of adenovirus (see, e.g.,U.S. Pat. Nos. 9,410,129, 7,344,872, 7,256,036, 6,911,199, 6,740,525;Matthews, “Capsid-Incorporation of Antigens into Adenovirus CapsidProteins for a Vaccine Approach,” Mol Pharm, 8(1): 3-11 (2011)), as wellas regarding modifications of AAV, the skilled person can readily obtaina modified adenovirus that has a large payload protein or aCRISPR-protein, despite that heretofore it was not expected that such alarge protein could be provided on an adenovirus. And as to the virusesrelated to adenovirus mentioned herein, as well as to the virusesrelated to AAV mentioned herein, the teachings herein as to modifyingadenovirus and AAV, respectively, can be applied to those viruseswithout undue experimentation from this disclosure and the knowledge inthe art.

In another aspect, the invention provides a non-naturally occurring orengineered CRISPR protein associated with Adeno Associated Virus (AAV),e.g., an AAV comprising a CRISPR protein as a fusion, with or without alinker, to or with an AAV capsid protein such as VP1, VP2, and/or VP3;and, for shorthand purposes, such a non-naturally occurring orengineered CRISPR protein is herein termed a “AAV-CRISPR protein” Morein particular, modifying the knowledge in the art, e.g., Rybniker etal., “Incorporation of Antigens into Viral Capsids AugmentsImmunogenicity of Adeno-Associated Virus Vector-Based Vaccines,” JVirol. December 2012; 86(24): 13800-13804, Lux K, et al. 2005. Greenfluorescent protein-tagged adeno-associated virus particles allow thestudy of cytosolic and nuclear trafficking. J. Virol. 79:11776-11787,Munch R C, et al. 2012. “Displaying high-affinity ligands onadeno-associated viral vectors enables tumor cell-specific and safe genetransfer.” Mol. Ther. [Epub ahead of print.] doi:10.1038/mt.2012.186 andWarrington K H, Jr, et al. 2004. Adeno-associated virus type 2 VP2capsid protein is nonessential and can tolerate large peptide insertionsat its N terminus. J. Virol. 78:6595-6609, each incorporated herein byreference, one can obtain a modified AAV capsid of the invention. Itwill be understood by those skilled in the art that the modificationsdescribed herein if inserted into the AAV cap gene may result inmodifications in the VP1, VP2 and/or VP3 capsid subunits. Alternatively,the capsid subunits can be expressed independently to achievemodification in only one or two of the capsid subunits (VP1, VP2, VP3,VP1+VP2, VP1+VP3, or VP2+VP3). One can modify the cap gene to haveexpressed at a desired location a non-capsid protein advantageously alarge payload protein, such as a CRISPR-protein. Likewise, these can befusions, with the protein, e.g., large payload protein such as aCRISPR-protein fused in a manner analogous to prior art fusions. See,e.g., US Patent Publication 20090215879; Nance et al., “Perspective onAdeno-Associated Virus Capsid Modification for Duchenne MuscularDystrophy Gene Therapy,” Hum Gene Ther. 26(12):786-800 (2015) anddocuments cited therein, incorporated herein by reference. The skilledperson, from this disclosure and the knowledge in the art can make anduse modified AAV or AAV capsid as in the herein invention, and throughthis disclosure one knows now that large payload proteins can be fusedto the AAV capsid. Applicants provide AAV capsid-CRISPR protein (e.g.,Cas, Cas9, dCas9, Cpf1, Cas13a, Cas13b) fusions and those AAV-capsidCRISPR protein (e.g., Cas, Cas9) fusions can be a recombinant AAV thatcontains nucleic acid molecule(s) encoding or providing CRISPR-Cas orCRISPR system or complex RNA guide(s), whereby the CRISPR protein (e.g.,Cas, Cas9) fusion delivers a CRISPR-Cas or CRISPR system complex (e.g.,the CRISPR protein or Cas or Cas9 or Cpf1 is provided by the fusion,e.g., VP1, VP2, pr VP3 fusion, and the guide RNA is provided by thecoding of the recombinant virus, whereby in vivo, in a cell, theCRISPR-Cas or CRISPR system is assembled from the nucleic acidmolecule(s) of the recombinant providing the guide RNA and the outersurface of the virus providing the CRISPR-Enzyme or Cas or Cas9. Such ascomplex may herein be termed an “AAV-CRISPR system” or an“AAV-CRISPR-Cas” or “AAV-CRISPR complex” or AAV-CRISPR-Cas complex.”Accordingly, the instant invention is also applicable to a virus in thegenus Dependoparvovirus or in the family Parvoviridae, for instance,AAV, or a virus of Amdoparvovirus, e.g., Carnivore amdoparvovirus 1, avirus of Aveparvovirus, e.g., Galliform aveparvovirus 1, a virus ofBocaparvovirus, e.g., Ungulate bocaparvovirus 1, a virus ofCopiparvovirus, e.g., Ungulate copiparvovirus 1, a virus ofDependoparvovirus, e.g., Adeno-associated dependoparvovirus A, a virusof Erythroparvovirus, e.g., Primate erythroparvovirus 1, a virus ofProtoparvovirus, e.g., Rodent protoparvovirus 1, a virus ofTetraparvovirus, e.g., Primate tetraparvovirus 1. Thus, a virus ofwithin the family Parvoviridae or the genus Dependoparvovirus or any ofthe other foregoing genera within Parvoviridae is contemplated as withinthe invention with discussion herein as to AAV applicable to such otherviruses.

In one aspect, the invention provides a non-naturally occurring orengineered composition comprising a CRISPR enzyme which is part of ortethered to a AAV capsid domain, i.e., VP1, VP2, or VP3 domain ofAdeno-Associated Virus (AAV) capsid. In some embodiments, part of ortethered to a AAV capsid domain includes associated with associated witha AAV capsid domain. In some embodiments, the CRISPR enzyme may be fusedto the AAV capsid domain. In some embodiments, the fusion may be to theN-terminal end of the AAV capsid domain. As such, in some embodiments,the C-terminal end of the CRISPR enzyme is fused to the N- terminal endof the AAV capsid domain. In some embodiments, an NLS and/or a linker(such as a GlySer linker) may be positioned between the C-terminal endof the CRISPR enzyme and the N- terminal end of the AAV capsid domain.In some embodiments, the fusion may be to the C-terminal end of the AAVcapsid domain. In some embodiments, this is not preferred due to thefact that the VP1, VP2 and VP3 domains of AAV are alternative splices ofthe same RNA and so a C-terminal fusion may affect all three domains. Insome embodiments, the AAV capsid domain is truncated. In someembodiments, some or all of the AAV capsid domain is removed. In someembodiments, some of the AAV capsid domain is removed and replaced witha linker (such as a GlySer linker), typically leaving the N- terminaland C-terminal ends of the AAV capsid domain intact, such as the first2, 5 or 10 amino acids. In this way, the internal (non-terminal) portionof the VP3 domain may be replaced with a linker. It is particularlypreferred that the linker is fused to the CRISPR protein. A branchedlinker may be used, with the CRISPR protein fused to the end of one ofthe braches. This allows for some degree of spatial separation betweenthe capsid and the CRISPR protein. In this way, the CRISPR protein ispart of (or fused to) the AAV capsid domain.

Alternatively, the CRISPR enzyme may be fused in frame within, i.e.internal to, the AAV capsid domain. Thus in some embodiments, the AAVcapsid domain again preferably retains its N- terminal and C-terminalends. In this case, a linker is preferred, in some embodiments, eitherat one or both ends of the CRISPR enzyme. In this way, the CRISPR enzymeis again part of (or fused to) the AAV capsid domain. In certainembodiments, the positioning of the CRISPR enzyme is such that theCRISPR enzyme is at the external surface of the viral capsid onceformed. In one aspect, the invention provides a non-naturally occurringor engineered composition comprising a CRISPR enzyme associated with aAAV capsid domain of Adeno-Associated Virus (AAV) capsid. Here,associated may mean in some embodiments fused, or in some embodimentsbound to, or in some embodiments tethered to. The CRISPR protein may, insome embodiments, be tethered to the VP1, VP2, or VP3 domain. This maybe via a connector protein or tethering system such as thebiotin-streptavidin system. In one example, a biotinylation sequence (15amino acids) could therefore be fused to the CRISPR protein. When afusion of the AAV capsid domain, especially the N- terminus of the AAVAAV capsid domain, with streptavidin is also provided, the two willtherefore associate with very high affinity. Thus, in some embodiments,provided is a composition or system comprising a CRISPR protein-biotinfusion and a streptavidin- AAV capsid domain arrangement, such as afusion. The CRISPR protein-biotin and streptavidin- AAV capsid domainforms a single complex when the two parts are brought together. NLSs mayalso be incorporated between the CRISPR protein and the biotin; and/orbetween the streptavidin and the AAV capsid domain.

An alternative tether may be to fuse or otherwise associate the AAVcapsid domain to an adaptor protein which binds to or recognizes to acorresponding RNA sequence or motif. In some embodiments, the adaptor isor comprises a binding protein which recognizes and binds (or is boundby) an RNA sequence specific for said binding protein. In someembodiments, a preferred example is the MS2 (see Konermann et al.December 2014, cited infra, incorporated herein by reference) bindingprotein which recognizes and binds (or is bound by) an RNA sequencespecific for the MS2 protein.

With the AAV capsid domain associated with the adaptor protein, theCRISPR protein may, in some embodiments, be tethered to the adaptorprotein of the AAV capsid domain. The CRISPR protein may, in someembodiments, be tethered to the adaptor protein of the AAV capsid domainvia the CRISPR enzyme being in a complex with a modified guide, seeKonermann et al. The modified guide is, in some embodiments, a sgRNA. Insome embodiments, the modified guide comprises a distinct RNA sequence;see, e.g., PCT/US14/70175, incorporated herein by reference.

In some embodiments, distinct RNA sequence is an aptamer. Thus,corresponding aptamer- adaptor protein systems are preferred. One ormore functional domains may also be associated with the adaptor protein.An example of a preferred arrangement would be: [AAV AAV capsid domain -adaptor protein]- [modified guide - CRISPR protein].

In certain embodiments, the positioning of the CRISPR protein is suchthat the CRISPR protein is at the internal surface of the viral capsidonce formed. In one aspect, the invention provides a non-naturallyoccurring or engineered composition comprising a CRISPR proteinassociated with an internal surface of an AAV capsid domain. Here again,associated may mean in some embodiments fused, or in some embodimentsbound to, or in some embodiments tethered to. The CRISPR protein may, insome embodiments, be tethered to the VP1, VP2, or VP3 domain such thatit locates to the internal surface of the viral capsid once formed. Thismay be via a connector protein or tethering system such as thebiotin-streptavidin system as described above.

When the CRISPR protein fusion is designed so as to position the CRISPRprotein at the internal surface of the capsid once formed, the CRISPRprotein will fill most or all of internal volume of the capsid.Alternatively the CRISPR protein may be modified or divided so as tooccupy a less of the capsid internal volume. Accordingly, in certainembodiments, the invention provides a CRISRP protein divided in twoportions, one portion comprises in one viral particle or capsid and thesecond portion comprised in a second viral particle or capsid. Incertain embodiments, by splitting the CRISPR protein in two portions,space is made available to link one or more heterologous domains to oneor both CRISPR protein portions.

Split CRISPR proteins are set forth herein and in documents incorporatedherein by reference in further detail herein. In certain embodiments,each part of a split CRISRP proteins are attached to a member of aspecific binding pair, and when bound with each other, the members ofthe specific binding pair maintain the parts of the CRISPR protein inproximity. In certain embodiments, each part of a split CRISPR proteinis associated with an inducible binding pair. An inducible binding pairis one which is capable of being switched “on” or “off” by a protein orsmall molecule that binds to both members of the inducible binding pair.In general, according to the invention, CRISPR proteins may preferablysplit between domains, leaving domains intact. Preferred, non-limitingexamples of such CRISPR proteins include, without limitation, Cas9,Cpf1, C2c2, Cas13a, Cas13b, and orthologues. Preferred, non-limitingexamples of split points include, with reference to SpCas9: a splitposition between 202A/203S; a split position between 255F/256D; a splitposition between 310E/311I; a split position between 534R/535K; a splitposition between 572E/573C; a split position between 713S/714G; a splitposition between 1003L/104E; a split position between 1054G/1055E; asplit position between 1114N/11155; a split position between1152K/1153S; a split position between 1245K/1246G; or a split between1098 and 1099.

In some embodiments, any AAV serotype is preferred. In some embodiments,the VP2 domain associated with the CRISPR enzyme is an AAV serotype 2VP2 domain. In some embodiments, the VP2 domain associated with theCRISPR enzyme is an AAV serotype 8 VP2 domain. The serotype can be amixed serotype as is known in the art.

The CRISPR enzyme may form part of a CRISPR-Cas system, which furthercomprises a guide RNA (sgRNA) comprising a guide sequence capable ofhybridizing to a target sequence in a genomic locus of interest in acell. In some embodiments, the functional CRISPR-Cas system binds to thetarget sequence. In some embodiments, the functional CRISPR-Cas systemmay edit the genomic locus to alter gene expression. In someembodiments, the functional CRISPR-Cas system may comprise furtherfunctional domains.

In some embodiments, the CRISPR enzyme is a Cpf1. In some embodiments,the CRISPR enzyme is an FnCpf1. In some embodiments, the CRISPR enzymeis an AsCpf1, although other orthologs are envisaged. FnCpf1 and AsCpf1are particularly preferred, in some embodiments.

In some embodiments, the CRISPR enzyme is external to the capsid orvirus particle. In the sense that it is not inside the capsid (envelopedor encompassed with the capsid), but is externally exposed so that itcan contact the target genomic DNA). In some embodiments, the CRISPRenzyme cleaves both strands of DNA to produce a double strand break(DSB). In some embodiments, the CRISPR enzyme is a nickase. In someembodiments, the CRISPR enzyme is a dual nickase. In some embodiments,the CRISPR enzyme is a deadCpf1. In some general embodiments, the CRISPRenzyme is associated with one or more functional domains. In some morespecific embodiments, the CRISPR enzyme is a deadCpf1 and is associatedwith one or more functional domains. In some embodiments, the CRISPRenzyme comprises a Rec2 or HD2 truncation. In some embodiments, theCRISPR enzyme is associated with the AAV VP2 domain by way of a fusionprotein. In some embodiments, the CRISPR enzyme is fused toDestabilization Domain (DD). In other words, the DD may be associatedwith the CRISPR enzyme by fusion with said CRISPR enzyme. The AAV canthen, by way of nucleic acid molecule(s) deliver the stabilizing ligand(or such can be otherwise delivered) In some embodiments, the enzyme maybe considered to be a modified CRISPR enzyme, wherein the CRISPR enzymeis fused to at least one destabilization domain (DD) and VP2. In someembodiments, the association may be considered to be a modification ofthe VP2 domain. Where reference is made herein to a modified VP2 domain,then this will be understood to include any association discussed hereinof the VP2 domain and the CRISPR enzyme. In some embodiments, the AAVVP2 domain may be associated (or tethered) to the CRISPR enzyme via aconnector protein, for example using a system such as thestreptavidin-biotin system. As such, provided is a fusion of a CRISPRenzyme with a connector protein specific for a high affinity ligand forthat connector, whereas the AAV VP2 domain is bound to said highaffinity ligand. For example, streptavidin may be the connector fused tothe CRISPR enzyme, while biotin may be bound to the AAV VP2 domain. Uponco-localization, the streptavidin will bind to the biotin, thusconnecting the CRISPR enzyme to the AAV VP2 domain. The reversearrangement is also possible. In some embodiments, a biotinylationsequence (15 amino acids) could therefore be fused to the AAV VP2domain, especially the N- terminus of the AAV VP2 domain. A fusion ofthe CRISPR enzyme with streptavidin is also preferred, in someembodiments. In some embodiments, the biotinylated AAV capsids withstreptavidin-CRISPR enzyme are assembled in vitro. This way the AAVcapsids should assemble in a straightforward manner and the CRISPRenzyme-streptavidin fusion can be added after assembly of the capsid. Inother embodiments a biotinylation sequence (15 amino acids) couldtherefore be fused to the CRISPR enzyme, together with a fusion of theAAV VP2 domain, especially the N- terminus of the AAV VP2 domain, withstreptavidin. For simplicity, a fusion of the CRISPR enzyme and the AAVVP2 domain is preferred in some embodiments. In some embodiments, thefusion may be to the N- terminal end of the CRISPR enzyme. In otherwords, in some embodiments, the AAV and CRISPR enzyme are associated viafusion. In some embodiments, the AAV and CRISPR enzyme are associatedvia fusion including a linker. Suitable linkers are discussed herein,but include Gly Ser linkers. Fusion to the N- term of AAV VP2 domain ispreferred, in some embodiments. In some embodiments, the CRISPR enzymecomprises at least one Nuclear Localization Signal (NLS). In an aspect,the present invention provides a polynucleotide encoding the presentCRISPR enzyme and associated AAV VP2 domain.

Viral delivery vectors, for example modified viral delivery vectors, arehereby provided. While the AAV may advantageously be a vehicle forproviding RNA of the CRISPR-Cas Complex or CRISPR system, another vectormay also deliver that RNA, and such other vectors are also hereindiscussed. In one aspect, the invention provides a non-naturallyoccurring modified AAV having a VP2-CRISPR enzyme capsid protein,wherein the CRISPR enzyme is part of or tethered to the VP2 domain. Insome preferred embodiments, the CRISPR enzyme is fused to the VP2 domainso that, in another aspect, the invention provides a non-naturallyoccurring modified AAV having a VP2-CRISPR enzyme fusion capsid protein.The following embodiments apply equally to either modified AAV aspect,unless otherwise apparent. Thus, reference herein to a VP2-CRISPR enzymecapsid protein may also include a VP2-CRISPR enzyme fusion capsidprotein. In some embodiments, the VP2-CRISPR enzyme capsid proteinfurther comprises a linker. In some embodiments, the VP2-CRISPR enzymecapsid protein further comprises a linker, whereby the VP2-CRISPR enzymeis distanced from the remainder of the AAV. In some embodiments, theVP2-CRISPR enzyme capsid protein further comprises at least one proteincomplex, e.g., CRISPR complex, such as CRISPR-Cpf1 complex guide RNAthat targets a particular DNA, TALE, etc. A CRISPR complex, such asCRISPR-Cas system comprising the VP2-CRISPR enzyme capsid protein and atleast one CRISPR complex, such as CRISPR-Cpf1 complex guide RNA thattargets a particular DNA, is also provided in one aspect. In general, insome embodiments, the AAV further comprises a repair template. It willbe appreciated that comprises here may mean encompassed thin the viralcapsid or that the virus encodes the comprised protein. In someembodiments, one or more, preferably two or more guide RNAs, may becomprised/encompassed within the AAV vector. Two may be preferred, insome embodiments, as it allows for multiplexing or dual nickaseapproaches. Particularly for multiplexing, two or more guides may beused. In fact, in some embodiments, three or more, four or more, five ormore, or even six or more guide RNAs may be comprised/encompassed withinthe AAV. More space has been freed up within the AAV by virtue of thefact that the AAV no longer needs to comprise/encompass the CRISPRenzyme. In each of these instances, a repair template may also beprovided comprised/encompassed within the AAV. In some embodiments, therepair template corresponds to or includes the DNA target.

In a further aspect, the present invention provides compositionscomprising the CRISPR enzyme and associated AAV VP2 domain or thepolynucleotides or vectors described herein. Also provides areCRISPR-Cas systems comprising guide RNAs.

Also provided is a method of treating a subject in need thereof,comprising inducing gene editing by transforming the subject with thepolynucleotide encoding the system or any of the present vectors. Asuitable repair template may also be provided, for example delivered bya vector comprising said repair template. In some embodiments, a singlevector provides the CRISPR enzyme through (association with the viralcapsid) and at least one of: guide RNA; and/or a repair template. Alsoprovided is a method of treating a subject in need thereof, comprisinginducing transcriptional activation or repression by transforming thesubject with the polynucleotide encoding the present system or any ofthe present vectors, wherein said polynucleotide or vector encodes orcomprises the catalytically inactive CRISPR enzyme and one or moreassociated functional domains. Compositions comprising the presentsystem for use in said method of treatment are also provided. A kit ofparts may be provided including such compositions. Use of the presentsystem in the manufacture of a medicament for such methods of treatmentare also provided.

Also provided is a pharmaceutical composition comprising the CRISPRenzyme which is part of or tethered to a VP2 domain of Adeno-AssociatedVirus (AAV) capsid; or the non-naturally occurring modified AAV; or apolynucleotide encoding them.

Also provided is a complex of the CRISPR enzyme with a guideRNA, such assgRNA. The complex may further include the target DNA.

A split CRISPR enzyme, most preferably Cpf1, approach may be used. Theso-called ‘split Cpf1’ approach Split Cpf1 allows for the following. TheCpf1 is split into two pieces and each of these are fused to one half ofa dimer. Upon dimerization, the two parts of the Cpf1 are broughttogether and the reconstituted Cpf1 has been shown to be functional.Thus, one part of the split Cpf1 may be associated with one VP2 domainand second part of the split Cpf1 may be associated with another VP2domain. The two VP2 domains may be in the same or different capsid. Inother words, the split parts of the Cpf1 could be on the same virusparticle or on different virus particles.

In some embodiments, one or more functional domains may be associatedwith or tethered to CRISPR enzyme and/or may be associated with ortethered to modified guides via adaptor proteins. These can be usedirrespective of the fact that the CRISPR enzyme may also be tethered toa virus outer protein or capsid or envelope, such as a VP2 domain or acapsid, via modified guides with aptamer RAN sequences that recognizecorrespond adaptor proteins.

In some embodiments, one or more functional domains comprise atranscriptional activator, repressor, a recombinase, a transposase, ahistone remodeler, a demethylase, a DNA methyltransferase, acryptochrome, a light inducible/controllable domain, a chemicallyinducible/controllable domain, an epigenetic modifying domain, or acombination thereof. Advantageously, the functional domain comprises anactivator, repressor or nuclease.

In some embodiments, a functional domain can have methylase activity,demethylase activity, transcription activation activity, transcriptionrepression activity, transcription release factor activity, histonemodification activity, RNA cleavage activity or nucleic acid bindingactivity, or activity that a domain identified herein has.

Examples of activators include P65, a tetramer of the herpes simplexactivation domain VP16, termed VP64, optimized use of VP64 foractivation through modification of both the sgRNA design and addition ofadditional helper molecules, MS2, P65 and HSF1 in the system called thesynergistic activation mediator (SAM) (Konermann et al, “Genome-scaletranscriptional activation by an engineered CRISPR-Cas9 complex,” Nature517(7536):583-8 (2015)); and examples of repressors include the KRAB(Kruppel-associated box) domain of Kox1 or SID domain (e.g. SID4X); andan example of a nuclease or nuclease domain suitable for a functionaldomain comprises Fok1.

Suitable functional domains for use in practice of the invention, suchas activators, repressors or nucleases are also discussed in documentsincorporated herein by reference, including the patents and patentpublications herein-cited and incorporated herein by reference regardinggeneral information on CRISPR-Cas Systems.

In some embodiments, the CRISPR enzyme comprises or consists essentiallyof or consists of a localization signal as, or as part of, the linkerbetween the CRISPR enzyme and the AAV capsid, e.g., VP2. HA or Flag tagsare also within the ambit of the invention as linkers as well as GlycineSerine linkers as short as GS up to (GGGGS)3. In this regard it ismentioned that tags that can be used in embodiments of the inventioninclude affinity tags, such as chitin binding protein (CBP), maltosebinding protein (MBP), glutathione-S-transferase (GST), poly(His) tag;solubilization tags such as thioredoxin (TRX) and poly(NANP), MBP, andGST; chromatography tags such as those consisting of polyanionic aminoacids, such as FLAG-tag; epitope tags such as V5-tag, Myc-tag, HA-tagand NE-tag; fluorescence tags, such as GFP and mCherry; protein tagsthat may allow specific enzymatic modification (such as biotinylation bybiotin ligase) or chemical modification (such as reaction withFlAsH-EDT2 for fluorescence imaging).

Also provided is a method of treating a subject, e.g, a subject in needthereof, comprising inducing gene editing by transforming the subjectwith the AAV-CRISPR enzyme advantageously encoding and expressing invivo the remaining portions of the CRISPR system (e.g., RNA, guides). Asuitable repair template may also be provided, for example delivered bya vector comprising said repair template. Also provided is a method oftreating a subject, e.g., a subject in need thereof, comprising inducingtranscriptional activation or repression by transforming the subjectwith the AAV-CRISPR enzyme advantageously encoding and expressing invivo the remaining portions of the CRISPR system (e.g., RNA, guides);advantageously in some embodiments the CRISPR enzyme is a catalyticallyinactive CRISPR enzyme and comprises one or more associated functionaldomains. Where any treatment is occurring ex vivo, for example in a cellculture, then it will be appreciated that the term ‘subject’ may bereplaced by the phrase “cell or cell culture.”

Compositions comprising the present system for use in said method oftreatment are also provided. A kit of parts may be provided includingsuch compositions. Use of the present system in the manufacture of amedicament for such methods of treatment are also provided. Use of thepresent system in screening is also provided by the present invention,e.g., gain of function screens. Cells which are artificially forced tooverexpress a gene are be able to down regulate the gene over time(re-establishing equilibrium) e.g. by negative feedback loops. By thetime the screen starts the unregulated gene might be reduced again.

In one aspect, the invention provides an engineered, non-naturallyoccurring CRISPR-Cas system comprising a AAV-Cas protein and a guide RNAthat targets a DNA molecule encoding a gene product in a cell, wherebythe guide RNA targets the DNA molecule encoding the gene product and theCas protein cleaves the DNA molecule encoding the gene product, wherebyexpression of the gene product is altered; and, wherein the Cas proteinand the guide RNA do not naturally occur together. The inventioncomprehends the guide RNA comprising a guide sequence fused to a tracrsequence. In an embodiment of the invention the Cas protein is a type IICRISPR-Cas protein and in a preferred embodiment the Cas protein is aCpf1 protein. The invention further comprehends the coding for the Casprotein being codon optimized for expression in a eukaryotic cell. In apreferred embodiment the eukaryotic cell is a mammalian cell and in amore preferred embodiment the mammalian cell is a human cell. In afurther embodiment of the invention, the expression of the gene productis decreased.

In another aspect, the invention provides an engineered, non-naturallyoccurring vector system comprising one or more vectors comprising afirst regulatory element operably linked to a CRISPR-Cas system guideRNA that targets a DNA molecule encoding a gene product and a AAV-Casprotein. The components may be located on same or different vectors ofthe system, or may be the same vector whereby the AAV-Cas protein alsodelivers the RNA of the CRISPR system. The guide RNA targets the DNAmolecule encoding the gene product in a cell and the AAV-Cas protein maycleaves the DNA molecule encoding the gene product (it may cleave one orboth strands or have substantially no nuclease activity), wherebyexpression of the gene product is altered; and, wherein the AAV-Casprotein and the guide RNA do not naturally occur together. The inventioncomprehends the guide RNA comprising a guide sequence fused to a tracrsequence. In an embodiment of the invention the AAV-Cas protein is atype II AAV-CRISPR-Cas protein and in a preferred embodiment the AAV-Casprotein is a AAV-Cpf1 protein. The invention further comprehends thecoding for the AAV-Cas protein being codon optimized for expression in aeukaryotic cell. In a preferred embodiment the eukaryotic cell is amammalian cell and in a more preferred embodiment the mammalian cell isa human cell. In a further embodiment of the invention, the expressionof the gene product is decreased.

In another aspect, the invention provides a method of expressing aneffector protein and guide RNA in a cell comprising introducing thevector according any of the vector delivery systems disclosed herein. Inan embodiment of the vector for delivering an effector protein, theminimal promoter is the Mecp2 promoter, tRNA promoter, or U6. In afurther embodiment, the minimal promoter is tissue specific.

The one or more polynucleotide molecules may be comprised within one ormore vectors. The invention comprehends such polynucleotide molecule(s),for instance such polynucleotide molecules operably configured toexpress the protein and/or the nucleic acid component(s), as well assuch vector(s).

In one aspect, the invention provides a vector system comprising one ormore vectors. In some embodiments, the system comprises: (a) a firstregulatory element operably linked to a tracr mate sequence and one ormore insertion sites for inserting one or more guide sequences upstreamof the tracr mate sequence, wherein when expressed, the guide sequencedirects sequence-specific binding of a AAV-CRISPR complex to a targetsequence in a eukaryotic cell, wherein the CRISPR complex comprises aAAV-CRISPR enzyme complexed with (1) the guide sequence that ishybridized to the target sequence, and (2) the tracr mate sequence thatis hybridized to the tracr sequence; and (b) said AAV-CRISPR enzymecomprising at least one nuclear localization sequence and/or at leastone NES; wherein components (a) and (b) are located on or in the same ordifferent vectors of the system. In some embodiments, component (a)further comprises the tracr sequence downstream of the tracr matesequence under the control of the first regulatory element. In someembodiments, component (a) further comprises two or more guide sequencesoperably linked to the first regulatory element, wherein when expressed,each of the two or more guide sequences direct sequence specific bindingof a AAV-CRISPR complex to a different target sequence in a eukaryoticcell. In some embodiments, the system comprises the tracr sequence underthe control of a third regulatory element, such as a polymerase IIIpromoter. In some embodiments, the tracr sequence exhibits at least 50%,60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along thelength of the tracr mate sequence when optimally aligned. Determiningoptimal alignment is within the purview of one of skill in the art. Forexample, there are publically and commercially available alignmentalgorithms and programs such as, but not limited to, ClustalW,Smith-Waterman in matlab, Bowtie, Geneious, Biopython and SeqMan. Insome embodiments, the AAV-CRISPR complex comprises one or more nuclearlocalization sequences of sufficient strength to drive accumulation ofsaid CRISPR complex in a detectable amount in the nucleus of aeukaryotic cell. Without wishing to be bound by theory, it is believedthat a nuclear localization sequence is not necessary for AAV-CRISPRcomplex activity in eukaryotes, but that including such sequencesenhances activity of the system, especially as to targeting nucleic acidmolecules in the nucleus and/or having molecules exit the nucleus. Insome embodiments, the AAV-CRISPR enzyme is a type II AAV-CRISPR systemenzyme. In some embodiments, the AAV-CRISPR enzyme is a AAV-Cpf1 enzyme.In some embodiments, the AAV-Cpf1 enzyme is derived from S. mutans, S.agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C.coli; N. salsuginis, N. tergarcus; S. auricularis, S. carnosus; N.meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C.botulinum, C. difficile, C. tetani, C. sordellii; Francisella tularensis1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10,Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC,Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, CandidatusMethanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237,Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonascrevioricanis 3, Prevotella disiens and Porphyromonas macacae (e.g., aCpf1 of one of these organisms modified to have or be associated with atleast one AAV), and may include further mutations or alterations or be achimeric Cpf1. The enzyme may be a AAV-Cpf1 homolog or ortholog. In someembodiments, the AAV-CRISPR enzyme is codon-optimized for expression ina eukaryotic cell. In some embodiments, the AAV-CRISPR enzyme directscleavage of one or two strands at the location of the target sequence.In some embodiments, the AAV-CRISPR enzyme lacks DNA strand cleavageactivity. In some embodiments, the first regulatory element is apolymerase III promoter. In some embodiments, the second regulatoryelement is a polymerase II promoter. In some embodiments, the guidesequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between10-30, or between 15-25, or between 15-20 nucleotides in length. Ingeneral, and throughout this specification, the term “vector” refers toa nucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses). Viralvectors also include polynucleotides carried by a virus for transfectioninto a host cell. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) areintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors.” Common expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). Again, the RNA of theCRISPR System, while advantageously delivered via the AAV-CRISPR enzymecan also be delivered separately, e.g. via a separate vector.

In one aspect, the invention provides an AAV-CRISPR enzyme comprisingone or more nuclear localization sequences and/or NES. In someembodiments, said AAV-CRISPR enzyme includes a regulatory element thatdrives transcription of component(s) of the CRISPR system (e.g., RNA,such as guide RNA and/or HR template nucleic acid molecule) in aeukaryotic cell such that said AAV-CRISPR enzyme delivers the CRISPRsystem accumulates in a detectable amount in the nucleus of theeukaryotic cell and/or is exported from the nucleus. In someembodiments, the regulatory element is a polymerase II promoter. In someembodiments, the AAV-CRISPR enzyme is a type II AAV-CRISPR systemenzyme. In some embodiments, the AAV-CRISPR enzyme is a AAV-Cpf1 enzyme.In some embodiments, the AAV-Cpf1 enzyme is derived from S. mutans, S.agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C.coli; N. salsuginis, N. tergarcus; S. auricularis, S. carnosus; N.meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C.botulinum, C. difficile, C. tetani, C. sordellii; Francisella tularensis1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10,Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC,Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, CandidatusMethanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237,Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonascrevioricanis 3, Prevotella disiens and Porphyromonas macacae (e.g.,Cpf1 modified to have or be associated with at least one AAV), and mayinclude further alteration or mutation of the Cpf1, and can be achimeric Cpf1. In some embodiments, the AAV-CRISPR enzyme iscodon-optimized for expression in a eukaryotic cell. In someembodiments, the AAV-CRISPR enzyme directs cleavage of one or twostrands at the location of the target sequence. In some embodiments, theAAV-CRISPR enzyme lacks or substantially DNA strand cleavage activity(e.g., no more than 5% nuclease activity as compared with a wild typeenzyme or enzyme not having the mutation or alteration that decreasesnuclease activity).

In one aspect, the invention provides a AAV-CRISPR enzyme comprising oneor more nuclear localization sequences of sufficient strength to driveaccumulation of said AAV-CRISPR enzyme in a detectable amount in thenucleus of a eukaryotic cell. In some embodiments, the AAV-CRISPR enzymeis a type II AAV-CRISPR system enzyme. In some embodiments, theAAV-CRISPR enzyme is a AAV-Cpf1 enzyme. In some embodiments, theAAV-Cpf1 enzyme is derived from S. mutans, S. agalactiae, S.equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C. coli; N.salsuginis, N. tergarcus; S. auricularis, S. carnosus; N. meningitides,N. gonorrhoeae; L. monocytogenes, L. ivanovii; C. botulinum, C.difficile, C. tetani, C. sordellii; Francisella tularensis 1, Prevotellaalbensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10,Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC,Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, CandidatusMethanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237,Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonascrevioricanis 3, Prevotella disiens and Porphyromonas macacae (e.g.,Cpf1 modified to have or be associated with at least one AAV), and mayinclude further alteration or mutation of the Cpf1, and can be achimeric Cpf1. In some embodiments, the AAV-CRISPR enzyme iscodon-optimized for expression in a eukaryotic cell. In someembodiments, the AAV-CRISPR enzyme directs cleavage of one or twostrands at the location of the target sequence. In some embodiments, theAAV-CRISPR enzyme lacks or substantially DNA strand cleavage activity(e.g., no more than 5% nuclease activity as compared with a wild typeenzyme or enzyme not having the mutation or alteration that decreasesnuclease activity).

In one aspect, the invention provides a eukaryotic host cell comprising(a) a first regulatory element operably linked to a tracr mate sequenceand one or more insertion sites for inserting one or more guidesequences upstream of the tracr mate sequence, wherein when expressed,the guide sequence directs sequence-specific binding of a AAV-CRISPRcomplex to a target sequence in a eukaryotic cell, wherein theAAV-CRISPR complex comprises a AAV-CRISPR enzyme complexed with (1) theguide sequence that is hybridized to the target sequence, and (2) thetracr mate sequence that is hybridized to the tracr sequence; and/or (b)a said AAV-CRISPR enzyme optionally comprising at least one nuclearlocalization sequence and/or NES. In some embodiments, the host cellcomprises components (a) and (b). In some embodiments, component (a),component (b), or components (a) and (b) are stably integrated into agenome of the host eukaryotic cell. In some embodiments, component (b)includes or contains component (a). In some embodiments, component (a)further comprises the tracr sequence downstream of the tracr matesequence under the control of the first regulatory element. In someembodiments, component (a) further comprises two or more guide sequencesoperably linked to the first regulatory element, wherein when expressed,each of the two or more guide sequences direct sequence specific bindingof a AAV-CRISPR complex to a different target sequence in a eukaryoticcell. In some embodiments, the eukaryotic host cell further comprises athird regulatory element, such as a polymerase III promoter, operablylinked to said tracr sequence. In some embodiments, the tracr sequenceexhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequencecomplementarity along the length of the tracr mate sequence whenoptimally aligned. In some embodiments, the AAV-CRISPR enzyme comprisesone or more nuclear localization sequences and/or nuclear exportsequences of sufficient strength to drive accumulation of said CRISPRenzyme in a detectable amount in of the nucleus of a eukaryotic cell. Insome embodiments, the AAV- CRISPR enzyme is a type II CRISPR systemenzyme. In some embodiments, the CRISPR enzyme is a Cpf1 enzyme. In someembodiments, the AAV-Cpf1 enzyme is derived from S. mutans, S.agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C.coli; N. salsuginis, N. tergarcus; S. auricularis, S. carnosus; N.meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C.botulinum, C. difficile, C. tetani, C. sordellii; Francisella tularensis1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10,Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC,Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, CandidatusMethanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237,Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonascrevioricanis 3, Prevotella disiens and Porphyromonas macacae (e.g.,Cpf1 modified to have or be associated with at least one AAV), and mayinclude further alteration or mutation of the Cpf1, and can be achimeric Cpf1. In some embodiments, the AAV-CRISPR enzyme iscodon-optimized for expression in a eukaryotic cell. In someembodiments, the AAV-CRISPR enzyme directs cleavage of one or twostrands at the location of the target sequence. In some embodiments, theAAV-CRISPR enzyme lacks or substantially DNA strand cleavage activity(e.g., no more than 5% nuclease activity as compared with a wild typeenzyme or enzyme not having the mutation or alteration that decreasesnuclease activity). In some embodiments, the first regulatory element isa polymerase III promoter. In some embodiments, the second regulatoryelement is a polymerase II promoter. In some embodiments, the guidesequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between10-30, or between 15-25, or between 15-20 nucleotides in length. In anaspect, the invention provides a non-human eukaryotic organism;preferably a multicellular eukaryotic organism, comprising a eukaryotichost cell according to any of the described embodiments. In otheraspects, the invention provides a eukaryotic organism; preferably amulticellular eukaryotic organism, comprising a eukaryotic host cellaccording to any of the described embodiments. The organism in someembodiments of these aspects may be an animal; for example a mammal.Also, the organism may be an arthropod such as an insect. The organismalso may be a plant. Further, the organism may be a fungus.Advantageously the organism is a host of AAV.

In one aspect, the invention provides a kit comprising one or more ofthe components described herein. In some embodiments, the kit comprisesa vector system and instructions for using the kit. In some embodiments,the vector system comprises (a) a first regulatory element operablylinked to a tracr mate sequence and one or more insertion sites forinserting one or more guide sequences upstream of the tracr matesequence, wherein when expressed, the guide sequence directssequence-specific binding of a CRISPR complex to a target sequence in aeukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzymecomplexed with (1) the guide sequence that is hybridized to the targetsequence, and (2) the tracr mate sequence that is hybridized to thetracr sequence; and/or (b) said AAV-CRISPR enzyme optionally comprisinga nuclear localization sequence. In some embodiments, the kit comprisescomponents (a) and (b) located on or in the same or different vectors ofthe system, e.g., (a) can be contained in (b). In some embodiments,component (a) further comprises the tracr sequence downstream of thetracr mate sequence under the control of the first regulatory element.In some embodiments, component (a) further comprises two or more guidesequences operably linked to the first regulatory element, wherein whenexpressed, each of the two or more guide sequences direct sequencespecific binding of a CRISPR complex to a different target sequence in aeukaryotic cell. In some embodiments, the system further comprises athird regulatory element, such as a polymerase III promoter, operablylinked to said tracr sequence. In some embodiments, the tracr sequenceexhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequencecomplementarity along the length of the tracr mate sequence whenoptimally aligned. In some embodiments, the CRISPR enzyme comprises oneor more nuclear localization sequences of sufficient strength to driveaccumulation of said CRISPR enzyme in a detectable amount in the nucleusof a eukaryotic cell. In some embodiments, the CRISPR enzyme is a typeII CRISPR system enzyme. In some embodiments, the CRISPR enzyme is aCpf1 enzyme. In some embodiments, the Cpf1 enzyme is derived from S.mutans, S. agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C.jejuni, C. coli; N. salsuginis, N. tergarcus; S. auricularis, S.carnosus; N. meningitides, N. gonorrhoeae; L. monocytogenes, L.ivanovii; C. botulinum, C. difficile, C. tetani, C. sordellii;Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacteriumMC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacteriumGW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithellasp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020,Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxellabovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006,Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonasmacacae (e.g., Cpf1 modified to have or be associated with at least oneAAV), and may include further alteration or mutation of the Cpf1, andcan be a chimeric Cpf1. In some embodiments, the coding for theAAV-CRISPR enzyme is codon-optimized for expression in a eukaryoticcell. In some embodiments, the AAV-CRISPR enzyme directs cleavage of oneor two strands at the location of the target sequence. In someembodiments, the AAV-CRISPR enzyme lacks or substantially DNA strandcleavage activity (e.g., no more than 5% nuclease activity as comparedwith a wild type enzyme or enzyme not having the mutation or alterationthat decreases nuclease activity). In some embodiments, the firstregulatory element is a polymerase III promoter. In some embodiments,the second regulatory element is a polymerase II promoter. In someembodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25nucleotides, or between 10-30, or between 15-25, or between 15-20nucleotides in length.

In one aspect, the invention provides a method of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a AAV-CRISPR complex to bind to the targetpolynucleotide, e.g., to effect cleavage of said target polynucleotide,thereby modifying the target polynucleotide, wherein the AAV-CRISPRcomplex comprises a AAV-CRISPR enzyme complexed with a guide sequencehybridized to a target sequence within said target polynucleotide,wherein said guide sequence is linked to a tracr mate sequence which inturn hybridizes to a tracr sequence. In some embodiments, said cleavagecomprises cleaving one or two strands at the location of the targetsequence by said AAV-CRISPR enzyme. In some embodiments, said cleavageresults in decreased transcription of a target gene. In someembodiments, the method further comprises repairing said cleaved targetpolynucleotide by homologous recombination with an exogenous templatepolynucleotide, wherein said repair results in a mutation comprising aninsertion, deletion, or substitution of one or more nucleotides of saidtarget polynucleotide. In some embodiments, said mutation results in oneor more amino acid changes in a protein expressed from a gene comprisingthe target sequence. In some embodiments, the method further comprisesdelivering one or more vectors to said eukaryotic cell, wherein one ormore vectors comprise the AAV-CRISPR enzyme and one or more vectorsdrive expression of one or more of: the guide sequence linked to thetracr mate sequence, and the tracr sequence. In some embodiments, saidAAV-CRISPR enzyme drive expression of one or more of: the guide sequencelinked to the tracr mate sequence, and the tracr sequence. In someembodiments such AAV-CRISPR enzyme are delivered to the eukaryotic cellin a subject. In some embodiments, said modifying takes place in saideukaryotic cell in a cell culture. In some embodiments, the methodfurther comprises isolating said eukaryotic cell from a subject prior tosaid modifying. In some embodiments, the method further comprisesreturning said eukaryotic cell and/or cells derived therefrom to saidsubject.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a AAV-CRISPR complex to bind to thepolynucleotide such that said binding results in increased or decreasedexpression of said polynucleotide; wherein the AAV-CRISPR complexcomprises a AAV-CRISPR enzyme complexed with a guide sequence hybridizedto a target sequence within said polynucleotide, wherein said guidesequence is linked to a tracr mate sequence which in turn hybridizes toa tracr sequence. In some embodiments, the method further comprisesdelivering one or more vectors to said eukaryotic cells, wherein the oneor more vectors are the AAV-CRISPR enzyme and/or drive expression of oneor more of: the guide sequence linked to the tracr mate sequence, andthe tracr sequence.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a mutated disease gene. In some embodiments,a disease gene is any gene associated an increase in the risk of havingor developing a disease. In some embodiments, the method comprises (a)introducing one or more vectors into a eukaryotic cell, wherein the oneor more vectors comprise the AAV-CRISPR enzyme and/or drive expressionof one or more of: a guide sequence linked to a tracr mate sequence, anda tracr sequence; and (b) allowing a AAV-CRISPR complex to bind to atarget polynucleotide, e.g., to effect cleavage of the targetpolynucleotide within said disease gene, wherein the AAV-CRISPR complexcomprises the AAV-CRISPR enzyme complexed with (1) the guide sequencethat is hybridized to the target sequence within the targetpolynucleotide, and (2) the tracr mate sequence that is hybridized tothe tracr sequence, thereby generating a model eukaryotic cellcomprising a mutated disease gene. Thus, in some embodiments theAAV-CRISPR enzyme contains nucleic acid molecules for and drivesexpression of one or more of: a guide sequence linked to a tracr matesequence, and a tracr sequence and/or a Homologous Recombinationtemplate and/or a stabilizing ligand if the CRISPR enzyme has adestabilization domain. In some embodiments, said cleavage comprisescleaving one or two strands at the location of the target sequence bysaid AAV-CRISPR enzyme. In some embodiments, said cleavage results indecreased transcription of a target gene. In some embodiments, themethod further comprises repairing said cleaved target polynucleotide byhomologous recombination with an exogenous template polynucleotide,wherein said repair results in a mutation comprising an insertion,deletion, or substitution of one or more nucleotides of said targetpolynucleotide. In some embodiments, said mutation results in one ormore amino acid changes in a protein expression from a gene comprisingthe target sequence.

In one aspect, the invention provides a method for developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. In some embodiments, a disease gene isany gene associated an increase in the risk of having or developing adisease. In some embodiments, the method comprises (a) contacting a testcompound with a model cell of any one of the described embodiments; and(b) detecting a change in a readout that is indicative of a reduction oran augmentation of a cell signaling event associated with said mutationin said disease gene, thereby developing said biologically active agentthat modulates said cell signaling event associated with said diseasegene.

In one aspect, the invention provides a recombinant polynucleotidecomprising a guide sequence upstream of a tracr mate sequence, whereinthe guide sequence when expressed directs sequence-specific binding of aAAV-CRISPR complex to a corresponding target sequence present in aeukaryotic cell. The polynucleotide can be carried within and expressedin vivo from the AAV-CRISPR enzyme. In some embodiments, the targetsequence is a viral sequence present in a eukaryotic cell. In someembodiments, the target sequence is a proto-oncogene or an oncogene.

In one aspect the invention provides for a method of selecting one ormore cell(s) by introducing one or more mutations in a gene in the oneor more cell (s), the method comprising: introducing one or more vectorsinto the cell (s), wherein the one or more vectors comprise a AAV-CRISPRenzyme and/or drive expression of one or more of: a guide sequencelinked to a tracr mate sequence, a tracr sequence, and an editingtemplate; wherein, for example that which is being expressed is withinand expressed in vivo by the AAV-CRISPR enzyme and/or the editingtemplate comprises the one or more mutations that abolish AAV-CRISPRenzyme cleavage; allowing homologous recombination of the editingtemplate with the target polynucleotide in the cell(s) to be selected;allowing a CRISPR complex to bind to a target polynucleotide to effectcleavage of the target polynucleotide within said gene, wherein theAAV-CRISPR complex comprises the AAV-CRISPR enzyme complexed with (1)the guide sequence that is hybridized to the target sequence within thetarget polynucleotide, and (2) the tracr mate sequence that ishybridized to the tracr sequence, wherein binding of the AAV-CRISPRcomplex to the target polynucleotide induces cell death, therebyallowing one or more cell(s) in which one or more mutations have beenintroduced to be selected. In a preferred embodiment, the AAV-CRISPRenzyme is AAV-Cpf1. In another aspect of the invention the cell to beselected may be a eukaryotic cell. Aspects of the invention allow forselection of specific cells without requiring a selection marker or atwo-step process that may include a counter-selection system. Thecell(s) may be prokaryotic or eukaryotic cells.

With respect to mutations of the AAV-CRISPR enzyme, mutations may bemade at any or all residues corresponding to positions 908, 993, and1263 with reference to amino acid position numbering of AsCpf1 (whichmay be ascertained for instance by standard sequence comparison tools),or 917 and 1006 with reference to amino acid numbering of FnCpf1, or832, 925, 947, 1180 with reference to amino acid position numbering ofLbCpf1. In particular, any or all of the following mutations arepreferred in AsCpf1: D908A, E993A, and D1263; in FnCpf1: D917A andH1006A; in LbCpf1: D832A, E925A, D947A, and D1180A; as well asconservative substitution for any of the replacement amino acids is alsoenvisaged. In an aspect the invention provides as to any or each or allembodiments herein-discussed wherein the AAV-CRISPR enzyme comprises atleast one or more, or at least two or more mutations, wherein the atleast one or more mutation or the at least two or more mutations is asto D908, E993, or D1263 according to AsCpf1 protein, e.g., D908A, E993A,or D1263 as to AsCpf1, or D917 or H1006 according to FnCpf1, e.g., D917Aor H1006A as to FnCpf1, or D832, E925, D947, or D1180 according toLbCpf1, e.g., D832A, E925A, D947A, or D1180A as to LbCpf1, or anycorresponding mutation(s) in a Cpf1 of an ortholog to As or Fn or Lb, orthe CRISPR enzyme comprises at least one mutation wherein at leastD908A, E993A, or D1263 as to AsCpf1 or D917A or H1006A as to FnCpf1 orD832A, E925A, D947A, or D1180A as to LbCpf1 is mutated; or anycorresponding mutation(s) in a Cpf1 of an ortholog to As protein or Fnprotein or Lb protein.

Aspects of the invention encompass a non-naturally occurring orengineered composition that may comprise a guide RNA (sgRNA) comprisinga guide sequence capable of hybridizing to a target sequence in agenomic locus of interest in a cell and a AAV-CRISPR enzyme that maycomprise at least one or more nuclear localization sequences, whereinthe AAV-CRISPR enzyme comprises one or two or more mutations, such thatthe enzyme has altered or diminished nuclease activity compared with thewild type enzyme, wherein at least one loop of the sgRNA is modified bythe insertion of distinct RNA sequence(s) that bind to one or moreadaptor proteins, and wherein the adaptor protein further recruits oneor more heterologous functional domains. In an embodiment of theinvention the AAV-CRISPR enzyme comprises one or two or more mutationsin a residue selected from the group comprising, consisting essentiallyof, or consisting of D908, E993, or D1263 according to AsCpf1 protein;D917 or H1006 according to FnCpf1; or D832, E925, D947, or D1180according to LbCpf1. In a further embodiment the AAV-CRISPR enzymecomprises one or two or more mutations selected from the groupcomprising D908A, E993A, or D1263 as to AsCpf1; D917A or H1006A as toFnCpf1; or D832A, E925A, D947A, or D1180A as to LbCpf1. In anotherembodiment, the functional domain comprise, consist essentially of atranscriptional activation domain, e.g., VP64. In another embodiment,the functional domain comprise, consist essentially of a transcriptionalrepressor domain, e.g., KRAB domain, SID domain or a SID4×domain. Inembodiments of the invention, the one or more heterologous functionaldomains have one or more activities selected from the group comprising,consisting essentially of, or consisting of methylase activity,demethylase activity, transcription activation activity, transcriptionrepression activity, transcription release factor activity, histonemodification activity, RNA cleavage activity and nucleic acid bindingactivity. In further embodiments of the invention the cell is aeukaryotic cell or a mammalian cell or a human cell. In furtherembodiments, the adaptor protein is selected from the group comprising,consisting essentially of, or consisting of MS2, PP7, Qβ, F2, GA, fr,JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI,ID2, NL95, TW19, AP205, #Cb5, #Cb8r, #Cbl2r, #Cb23r, 7s, PRR1. Inanother embodiment, the at least one loop of the sgRNA is tetraloopand/or loop2. An aspect of the invention encompasses methods ofmodifying a genomic locus of interest to change gene expression in acell by introducing into the cell any of the compositions describedherein. An aspect of the invention is that the above elements arecomprised in a single composition or comprised in individualcompositions, e.g., the AAV-CRISPR enzyme delivers the enzyme asdiscussed as well as the guide. These compositions may advantageously beapplied to a host to elicit a functional effect on the genomic level. Ingeneral, the sgRNA are modified in a manner that provides specificbinding sites (e.g., aptamers) for adapter proteins comprising one ormore functional domains (e.g., via fusion protein) to bind to. Themodified sgRNA are modified such that once the sgRNA forms a AAV-CRISPRcomplex (i.e. AAV-CRISPR enzyme binding to sgRNA and target) the adapterproteins bind and, the functional domain on the adapter protein ispositioned in a spatial orientation which is advantageous for theattributed function to be effective. For example, if the functionaldomain comprise, consist essentially of a transcription activator (e.g.,VP64 or p65), the transcription activator is placed in a spatialorientation which allows it to affect the transcription of the target.Likewise, a transcription repressor will be advantageously positioned toaffect the transcription of the target and a nuclease (e.g., Fok1) willbe advantageously positioned to cleave or partially cleave the target.Again, the AAV-CRISPR enzyme can deliver both the enzyme and themodified guide. The skilled person will understand that modifications tothe sgRNA which allow for binding of the adapter+functional domain butnot proper positioning of the adapter+functional domain (e.g., due tosteric hindrance within the three dimensional structure of the CRISPRcomplex) are modifications which are not intended. The one or moremodified sgRNA may be modified at the tetra loop, the stem loop 1, stemloop 2, or stem loop 3, as described herein, preferably at either thetetra loop or stem loop 2, and most preferably at both the tetra loopand stem loop 2.

As explained herein the functional domains may be, for example, one ormore domains from the group comprising, consisting essentially of, orconsisting of methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity, DNA cleavage activity, nucleic acid binding activity, andmolecular switches (e.g., light inducible). In some cases it isadvantageous that additionally at least one NLS is provided. In someinstances, it is advantageous to position the NLS at the N terminus.When more than one functional domain is included, the functional domainsmay be the same or different.

The sgRNA may be designed to include multiple binding recognition sites(e.g., aptamers) specific to the same or different adapter protein. ThesgRNA may be designed to bind to the promoter region −1000 −+1 nucleicacids upstream of the transcription start site (i.e. TSS), preferably−200 nucleic acids. This positioning improves functional domains whichaffect gene activation (e.g., transcription activators) or geneinhibition (e.g., transcription repressors). The modified sgRNA may beone or more modified sgRNAs targeted to one or more target loci (e.g.,at least 1 sgRNA, at least 2 sgRNA, at least 5 sgRNA, at least 10 sgRNA,at least 20 sgRNA, at least 30 sg RNA, at least 50 sgRNA) comprised in acomposition.

Further, the AAV-CRISPR enzyme with diminished nuclease activity is mosteffective when the nuclease activity is inactivated (e.g., nucleaseinactivation of at least 70%, at least 80%, at least 90%, at least 95%,at least 97%, or 100% as compared with the wild type enzyme; or to putin another way, a AAV-Cpf1 enzyme or AAV-CRISPR enzyme havingadvantageously about 0% of the nuclease activity of the non-mutated orwild type Cpf1 enzyme or CRISPR enzyme, or no more than about 3% orabout 5% or about 10% of the nuclease activity of the non-mutated orwild type Cpf1 enzyme or CRISPR enzyme). This is possible by introducingmutations into the RuvC and HNH nuclease domains of the AsCpf1 andorthologs thereof. For example utilizing mutations in a residue selectedfrom the group comprising, consisting essentially of, or consisting ofD908, E993, or D1263 according to AsCpf1 protein; D917 or H1006according to FnCpf1; or D832, E925, D947, or D1180 according to LbCpf1,and more preferably introducing one or more of the mutations selectedfrom the group comprising, consisting essentially of, or consisting ofD908A, E993A, or D1263 as to AsCpf1; D917A or H1006A as to FnCpf1; orD832A, E925A, D947A, or D1180A as to LbCpf1. The inactivated CRISPRenzyme may have associated (e.g., via fusion protein) one or morefunctional domains, e.g., at least one destabilizing domain; or, forinstance like those as described herein for the modified sgRNA adaptorproteins, including for example, one or more domains from the groupcomprising, consisting essentially of, or consisting of methylaseactivity, demethylase activity, transcription activation activity,transcription repression activity, transcription release factoractivity, histone modification activity, RNA cleavage activity, DNAcleavage activity, nucleic acid binding activity, and molecular switches(e.g., light inducible). Preferred domains are Fok1, VP64, P65, HSF1,MyoD1. In the event that Fok1 is provided, it is advantageous thatmultiple Fok1 functional domains are provided to allow for a functionaldimer and that sgRNAs are designed to provide proper spacing forfunctional use (Fok1) as specifically described in Tsai et al. NatureBiotechnology, Vol. 32, Number 6, June 2014). The adaptor protein mayutilize known linkers to attach such functional domains. In some casesit is advantageous that additionally at least one NLS is provided. Insome instances, it is advantageous to position the NLS at the Nterminus. When more than one functional domain is included, thefunctional domains may be the same or different. In general, thepositioning of the one or more functional domain on the inactivatedAAV-CRISPR enzyme is one which allows for correct spatial orientationfor the functional domain to affect the target with the attributedfunctional effect. For example, if the functional domain is atranscription activator (e.g., VP64 or p65), the transcription activatoris placed in a spatial orientation which allows it to affect thetranscription of the target. Likewise, a transcription repressor will beadvantageously positioned to affect the transcription of the target, anda nuclease (e.g., Fok1) will be advantageously positioned to cleave orpartially cleave the target. This may include positions other than theN—/C-terminus of the AAV-CRISPR enzyme. Positioning the functionaldomain in the Rec1 domain, the Rec2 domain, the HNH domain, or the PIdomain of the AsCpf1 protein or any ortholog corresponding to thesedomains is advantageous; and again, it is mentioned that the functionaldomain can be a DD. Positioning of the functional domains to the Recdomain or the Rec2 domain, of the AsCpf1 protein or any orthologcorresponding to these domains, in some instances may be preferred. Fok1functional domain may be attached at the N terminus. When more than onefunctional domain is included, the functional domains may be the same ordifferent.

An adaptor protein may be any number of proteins that binds to anaptamer or recognition site introduced into the modified sgRNA and whichallows proper positioning of one or more functional domains, once thesgRNA has been incorporated into the AAV-CRISPR complex, to affect thetarget with the attributed function. As explained in detail in thisapplication such may be coat proteins, preferably bacteriophage coatproteins. The functional domains associated with such adaptor proteins(e.g., in the form of fusion protein) may include, for example, one ormore domains from the group comprising, consisting essentially of, orconsisting of methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity, DNA cleavage activity, nucleic acid binding activity, andmolecular switches (e.g., light inducible). Preferred domains are Fok1,VP64, P65, HSF1, MyoD1. In the event that the functional domain is atranscription activator or transcription repressor it is advantageousthat additionally at least an NLS is provided and preferably at the Nterminus. When more than one functional domain is included, thefunctional domains may be the same or different. The adaptor protein mayutilize known linkers to attach such functional domains. Such linkersmay be used to associate the AAV (e.g., capsid or VP2) with the CRISPRenzyme or have the CRISPR enzyme comprise the AAV (or vice versa).

Thus, sgRNA, e.g., modified sgRNA, the inactivated AAV-CRISPR enzyme(with or without functional domains), and the binding protein with oneor more functional domains, may each individually be comprised in acomposition and administered to a host individually or collectively.Alternatively, these components may be provided in a single compositionfor administration to a host, e.g., the AAV-CRISPR enzyme can deliverthe RNA or guide or sgRNA or modified sgRNA and/or other components ofthe CRISPR system. Administration to a host may be performed via viralvectors, advantageously using the AAV-CRISPR enzyme as the deliveryvehicle, although other vehicles can be used to deliver components otherthan the enzyme of the CRISPR system, and such viral vectors can be, forexample, lentiviral vector, adenoviral vector, AAV vector. Severalvariations are appropriate to elicit a genomic locus event, includingDNA cleavage, gene activation, or gene deactivation. Using the providedcompositions, the person skilled in the art can advantageously andspecifically target single or multiple loci with the same or differentfunctional domains to elicit one or more genomic locus events. Thecompositions may be applied in a wide variety of methods for screeningin libraries in cells and functional modeling in vivo (e.g., geneactivation of lincRNA and identification of function; gain-of-functionmodeling; loss-of-function modeling; the use the compositions of theinvention to establish cell lines and transgenic animals foroptimization and screening purposes).

In an aspect, the invention provides a particle delivery system or thedelivery system or the virus particle of any one of any one of the aboveembodiments or the cell of any one of the above embodiments for use inmedicine or in therapy; or for use in a method of modifying an organismor a non-human organism by manipulation of a target sequence in agenomic locus associated with a disease or disorder; or for use in amethod of treating or inhibiting a condition caused by one or moremutations in a genetic locus associated with a disease in a eukaryoticorganism or a non-human organism.; or for use in in vitro, ex vivo or invivo gene or genome editing; or for use in in vitro, ex vivo or in vivogene therapy.

In an aspect, the invention provides a pharmaceutical compositioncomprising the particle delivery system or the delivery system or thevirus particle of any one of the above embodiment or the cell of any oneof the above embodiment.

Vaccines

In certain example embodiments, the agent may be a vaccine. Therapeuticvaccines represent a viable option for active immunotherapy of cancersthat aim to treat disease by using a patient's own immune system. Insome embodiments, these include autologous tumor cell vaccines that areprepared using patient-derived tumor cells. These are typicallyirradiated, combined with an immunostimulatory adjuvant, and thenadministered to the individual from whom the tumor cells were isolated.Autologous tumor cells may be modified to confer higherimmunostimulatory characteristics.

In some embodiments, allogeneic tumor cell vaccines, which typicallycontain two or three established human tumor cell lines, may be used toovercome many limitations of autologous tumor cell vaccines. Theseinclude limitless sources of tumor antigens, standardized andlarge-scale vaccine production, reliable analysis of clinical outcomes,easy manipulation for expression of immunostimulatory molecules andcost-effectiveness.

Dendritic cells are potent professional antigen-presenting cells thatact as sentinels at peripheral tissues where they uptake, process andpresent pathogen- or host-derived antigenic peptides to naïve Tlymphocytes at the lymphoid organs in the context of majorhistocompatibility molecules. Many cancer immunotherapeutic strategiestarget dendritic cells directly or indirectly for the induction ofantigen-specific immune responses. Preparation of dendric cell vaccinescan be achieved by loading tumor-associated antigens to patients'autologous dendritic cells that are simultaneously treated withadjuvants. These antigen-loaded, ex vivo matured dendritic cells areadministered back into patients to induce anti-tumor immunity. Antigensutilized for this purpose include tumor-derived proteins or peptides,whole tumor cells, DNA/RNA/virus, or fusion of tumor cells and dendriticcells.

Other suitable cancer vaccines known to those of skill in the artinclude, but are not necessarily limited to, protein/peptide-basedcancer vaccines using tumor-associated antigens as therapeutic targets(neoantigen vaccines), genetic vaccines such as DNA, RNA, or viral basedvaccines, or neoantigen vaccines as described in Guo et al. (Adv CancerRes 119:421-475; 2013).

In some embodiments, the agent is administered in a combinationtreatment regimen using a neoantigen vaccine.

Combination Treatment Regimens

In some embodiments, the agent may be administered in a combinationtreatment regimen such as one including checkpoint blockade therapy,vaccines, targeted therapies, radiation therapy, chemotherapy, and/oradoptive cell therapy (ACT) as described in more detail below.

Checkpoint Blockade Therapy

“Anti-immune checkpoint” or “immune checkpoint inhibitor or “immunecheckpoint blockade” therapy refers to the use of agents that inhibitimmune checkpoint nucleic acids and/or proteins. Immune checkpointsshare the common function of providing inhibitory signals that suppressimmune response and inhibition of one or more immune checkpoints canblock or otherwise neutralize inhibitory signaling to thereby upregulatean immune response in order to more efficaciously treat cancer.Exemplary agents useful for inhibiting immune checkpoints includeantibodies, small molecules, peptides, peptidomimetics, natural ligands,and derivatives of natural ligands, that can either bind and/orinactivate or inhibit immune checkpoint proteins, or fragments thereof;as well as RNA interference, antisense, nucleic acid aptamers, etc. thatcan downregulate the expression and/or activity of immune checkpointnucleic acids, or fragments thereof. Exemplary agents for upregulatingan immune response include antibodies against one or more immunecheckpoint proteins block the interaction between the proteins and itsnatural receptor(s); a non-activating form of one or more immunecheckpoint proteins (e.g., a dominant negative polypeptide); smallmolecules or peptides that block the interaction between one or moreimmune checkpoint proteins and its natural receptor(s); fusion proteins(e.g. the extracellular portion of an immune checkpoint inhibitionprotein fused to the Fc portion of an antibody or immunoglobulin) thatbind to its natural receptor(s); nucleic acid molecules that blockimmune checkpoint nucleic acid transcription or translation; and thelike. Such agents can directly block the interaction between the one ormore immune checkpoints and its natural receptor(s) (e.g., antibodies)to prevent inhibitory signaling and upregulate an immune response.Alternatively, agents can indirectly block the interaction between oneor more immune checkpoint proteins and its natural receptor(s) toprevent inhibitory signaling and upregulate an immune response. Forexample, a soluble version of an immune checkpoint protein ligand suchas a stabilized extracellular domain can bind to its receptor toindirectly reduce the effective concentration of the receptor to bind toan appropriate ligand. In one embodiment, anti-PD-1 antibodies,anti-PD-L1 antibodies, and/or anti-PD-L2 antibodies, either alone or incombination, are used to inhibit immune checkpoints. These embodimentsare also applicable to specific therapy against particular immunecheckpoints, such as the PD-1 pathway (e.g., anti-PD-1 pathway therapy,otherwise known as PD-1 pathway inhibitor therapy). Numerous immunecheckpoint inhibitors are known and publicly available including, forexample, Keytruda® (pembrolizumab; anti-PD-1 antibody), Opdivo®(nivolumab; anti-PD-1 antibody), Tecentriq® (atezolizumab; anti-PD-L1antibody), durvalumab (anti-PD-L1 antibody), and the like. Suchembodiments are equally applicable to the KLRB1 pathway, such asmodulating the interaction between KLRB1 and one or more natural bindingpartners, such as a CLEC2D.

In specific exemplary embodiments, the checkpoint blockade therapy mayinclude anti-PD-1, anti-CTLA4, anti-PDL1, anti-TIM-3 and/or anti-LAG3.

Adoptive Cell Therapy (Act)

In certain embodiments, immune cells are adoptively transferred to apatient for treatment of a disease. In certain embodiments, the immunecells are modified or modulated to enhance an immune response. Incertain embodiments, the transferred cells are modified or modulated todecrease expression or activity of KLRB1 or inactivate KLRB1. The cellsmay be further modified according to any embodiment described herein.For example, in addition to modulating the KLRB1 gene, cells foradoptive transfer may be further modified as described further herein(e.g., express a chimeric antigen receptor or TCR specific for a tumorantigen). As used herein, “ACT”, “adoptive cell therapy” and “adoptivecell transfer” may be used interchangeably. In certain embodiments,Adoptive cell therapy (ACT) can refer to the transfer of cells to apatient with the goal of transferring the functionality andcharacteristics into the new host by engraftment of the cells (see,e.g., Mettananda et al., Editing an a-globin enhancer in primary humanhematopoietic stem cells as a treatment for β-thalassemia, Nat Commun.2017 Sep. 4; 8(1):424). As used herein, the term “engraft” or“engraftment” refers to the process of cell incorporation into a tissueof interest in vivo through contact with existing cells of the tissue.Adoptive cell therapy (ACT) can refer to the transfer of cells, mostcommonly immune-derived cells, back into the same patient or into a newrecipient host with the goal of transferring the immunologicfunctionality and characteristics into the new host. If possible, use ofautologous cells helps the recipient by minimizing GVHD issues. Theadoptive transfer of autologous tumor infiltrating lymphocytes (TIL)(Besser et al., (2010) Clin. Cancer Res 16 (9) 2646-55; Dudley et al.,(2002) Science 298 (5594): 850-4; and Dudley et al., (2005) Journal ofClinical Oncology 23 (10): 2346-57.) or genetically re-directedperipheral blood mononuclear cells (Johnson et al., (2009) Blood 114(3): 535-46; and Morgan et al., (2006) Science 314(5796) 126-9) has beenused to successfully treat patients with advanced solid tumors,including melanoma and colorectal carcinoma, as well as patients withCD19-expressing hematologic malignancies (Kalos et al., (2011) ScienceTranslational Medicine 3 (95): 95ra73). In certain embodiments,allogenic cells immune cells are transferred (see, e.g., Ren et al.,(2017) Clin Cancer Res 23 (9) 2255-2266). As described further herein,allogenic cells can be edited to reduce alloreactivity and preventgraft-versus-host disease. Thus, use of allogenic cells allows for cellsto be obtained from healthy donors and prepared for use in patients asopposed to preparing autologous cells from a patient after diagnosis.

Aspects of the invention involve the adoptive transfer of immune systemcells, such as T cells, specific for selected antigens, such as tumorassociated antigens or tumor specific neoantigens (see, e.g., Maus etal., 2014, Adoptive Immunotherapy for Cancer or Viruses, Annual Reviewof Immunology, Vol. 32: 189-225; Rosenberg and Restifo, 2015, Adoptivecell transfer as personalized immunotherapy for human cancer, ScienceVol. 348 no. 6230 pp. 62-68; Restifo et al., 2015, Adoptiveimmunotherapy for cancer: harnessing the T cell response. Nat. Rev.Immunol. 12(4): 269-281; and Jenson and Riddell, 2014, Design andimplementation of adoptive therapy with chimeric antigenreceptor-modified T cells. Immunol Rev. 257(1): 127-144; and Rajasagi etal., 2014, Systematic identification of personal tumor-specificneoantigens in chronic lymphocytic leukemia. Blood. 2014 Jul. 17;124(3):453-62).

In certain embodiments, an antigen (such as a tumor antigen) to betargeted in adoptive cell therapy (such as particularly CAR or TCRT-cell therapy) of a disease (such as particularly of tumor or cancer)may be selected from a group consisting of: B cell maturation antigen(BCMA) (see, e.g., Friedman et al., Effective Targeting of MultipleBCMA-Expressing Hematological Malignancies by Anti-BCMA CAR T Cells, HumGene Ther. 2018 Mar. 8; Berdeja J G, et al. Durable clinical responsesin heavily pretreated patients with relapsed/refractory multiplemyeloma: updated results from a multicenter study of bb2121 anti-BcmaCAR T cell therapy. Blood. 2017; 130:740; and Mouhieddine and Ghobrial,Immunotherapy in Multiple Myeloma: The Era of CAR T Cell Therapy,Hematologist, May-June 2018, Volume 15, issue 3); PSA (prostate-specificantigen); prostate-specific membrane antigen (PSMA); PSCA (Prostate stemcell antigen); Tyrosine-protein kinase transmembrane receptor ROR1;fibroblast activation protein (FAP); Tumor-associated glycoprotein 72(TAG72); Carcinoembryonic antigen (CEA); Epithelial cell adhesionmolecule (EPCAM); Mesothelin; Human Epidermal growth factor Receptor 2(ERBB2 (Her2/neu)); Prostase; Prostatic acid phosphatase (PAP);elongation factor 2 mutant (ELF2M); Insulin-like growth factor 1receptor (IGF-1R); gplOO; BCR-ABL (breakpoint cluster region-Abelson);tyrosinase; New York esophageal squamous cell carcinoma 1 (NY-ESO—1);x-light chain, LAGE (L antigen); MAGE (melanoma antigen);Melanoma-associated antigen 1 (MAGE-A1); MAGE A3; MAGE A6; legumain;Human papillomavirus (HPV) E6; HPV E7; prostein; survivin; PCTA1(Galectin 8); Melan-A/MART-1; Ras mutant; TRP-1 (tyrosinase relatedprotein 1, or gp75); Tyrosinase-related Protein 2 (TRP2); TRP-2/INT2(TRP-2/intron 2); RAGE (renal antigen); receptor for advanced glycationend products 1 (RAGEl); Renal ubiquitous 1, 2 (RU1, RU2); intestinalcarboxyl esterase (iCE); Heat shock protein 70-2 (HSP70-2) mutant;thyroid stimulating hormone receptor (TSHR); CD123; CD171; CD19; CD20;CD22; CD26; CD30; CD33; CD44v7/8 (cluster of differentiation 44, exons7/8); CD53; CD92; CD100; CD148; CD150; CD200; CD261; CD262; CD362; CS-1(CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-likemolecule-1 (CLL-1); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (Tn Ag);Fms-Like Tyrosine Kinase 3 (FLT3); CD38; CD138; CD44v6; B7H3 (CD276);KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2);Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen(PSCA); Protease Serine 21 (PRSS21); vascular endothelial growth factorreceptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growthfactor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4(SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16 (MUC16);epidermal growth factor receptor (EGFR); epidermal growth factorreceptor variant III (EGFRvIII); neural cell adhesion molecule (NCAM);carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit,Beta Type, 9 (LMP2); ephrin type-A receptor 2 (EphA2); Ephrin B2;Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; high molecularweight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside(OAcGD2); Folate receptor alpha; Folate receptor beta; tumor endothelialmarker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R);claudin 6 (CLDN6); G protein-coupled receptor class C group 5, member D(GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a;anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1(PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH);mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2);Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3(ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2(OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumorprotein (WT1); ETS translocation-variant gene 6, located on chromosome12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A(XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); CT(cancer/testis (antigen)); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53;p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin Bi; Cyclin Di; v-myc avian myelocytomatosisviral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog FamilyMember C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor(Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma AntigenRecognized By T Cells-1 or 3 (SART1, SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint-1, -2, -3 or -4 (SSX1, SSX2, SSX3, SSX4);CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1(LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300molecule-like family member f (CD300LF); C-type lectin domain family 12member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyteantigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); mousedouble minute 2 homolog (MDM2); livin; alphafetoprotein (AFP);transmembrane activator and CAML Interactor (TACI); B-cell activatingfactor receptor (BAFF-R); V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog (KRAS); immunoglobulin lambda-like polypeptide 1 (IGLL1); 707-AP(707 alanine proline); ART-4 (adenocarcinoma antigen recognized by T4cells); BAGE (B antigen; b-catenin/m, b-catenin/mutated); CAMEL(CTL-recognized antigen on melanoma); CAPi (carcinoembryonic antigenpeptide 1); CASP-8 (caspase-8); CDC27m (cell-division cycle 27 mutated);CDK4/m (cycline-dependent kinase 4 mutated); Cyp-B (cyclophilin B); DAM(differentiation antigen melanoma); EGP-2 (epithelial glycoprotein 2);EGP-40 (epithelial glycoprotein 40); Erbb2, 3, 4 (erythroblasticleukemia viral oncogene homolog-2, -3, 4); FBP (folate binding protein);, fAchR (Fetal acetylcholine receptor); G250 (glycoprotein 250); GAGE (Gantigen); GnT-V (N-acetylglucosaminyltransferase V); HAGE (helicoseantigen); ULA-A (human leukocyte antigen-A); HST2 (human signet ringtumor 2); KIAA0205; KDR (kinase insert domain receptor); LDLR/FUT (lowdensity lipid receptor/GDP L-fucose: b-D-galactosidase 2-a-Lfucosyltransferase); L1CAM (L1 cell adhesion molecule); MC1R(melanocortin 1 receptor); Myosin/m (myosin mutated); MUM-1, -2, -3(melanoma ubiquitous mutated 1, 2, 3); NA88-A (NA cDNA clone of patientM88); KG2D (Natural killer group 2, member D) ligands; oncofetal antigen(h5T4); p190 minor bcr-abl (protein of 190KD bcr-abl); Pml/RARa(promyelocytic leukaemia/retinoic acid receptor a); PRAME(preferentially expressed antigen of melanoma); SAGE (sarcoma antigen);TEL/AML1 (translocation Ets-family leukemia/acute myeloid leukemia 1);TPI/m (triosephosphate isomerase mutated); CD70; and any combinationthereof.

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a tumor-specific antigen(TSA).

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a neoantigen.

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a tumor-associated antigen(TAA).

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a universal tumor antigen.In certain preferred embodiments, the universal tumor antigen isselected from the group consisting of: a human telomerase reversetranscriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2),cytochrome P450 1B 1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1),livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16(MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin(Dl), and any combinations thereof.

In certain embodiments, an antigen (such as a tumor antigen) to betargeted in adoptive cell therapy (such as particularly CAR or TCRT-cell therapy) of a disease (such as particularly of tumor or cancer)may be selected from a group consisting of: CD19, BCMA, CD70, CLL-1,MAGE A3, MAGE A6, HPV E6, HPV E7, WT1, CD22, CD171, ROR1, MUC16, andSSX2. In certain preferred embodiments, the antigen may be CD19. Forexample, CD19 may be targeted in hematologic malignancies, such as inlymphomas, more particularly in B-cell lymphomas, such as withoutlimitation in diffuse large B-cell lymphoma, primary mediastinal b-celllymphoma, transformed follicular lymphoma, marginal zone lymphoma,mantle cell lymphoma, acute lymphoblastic leukemia including adult andpediatric ALL, non-Hodgkin lymphoma, indolent non-Hodgkin lymphoma, orchronic lymphocytic leukemia. For example, BCMA may be targeted inmultiple myeloma or plasma cell leukemia (see, e.g., 2018 AmericanAssociation for Cancer Research (AACR) Annual meeting Poster: AllogeneicChimeric Antigen Receptor T Cells Targeting B Cell Maturation Antigen).For example, CLL1 may be targeted in acute myeloid leukemia. Forexample, MAGE A3, MAGE A6, SSX2, and/or KRAS may be targeted in solidtumors. For example, HPV E6 and/or HPV E7 may be targeted in cervicalcancer or head and neck cancer. For example, WT1 may be targeted inacute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronicmyeloid leukemia (CML), non-small cell lung cancer, breast, pancreatic,ovarian or colorectal cancers, or mesothelioma. For example, CD22 may betargeted in B cell malignancies, including non-Hodgkin lymphoma, diffuselarge B-cell lymphoma, or acute lymphoblastic leukemia. For example,CD171 may be targeted in neuroblastoma, glioblastoma, or lung,pancreatic, or ovarian cancers. For example, ROR1 may be targeted inROR1+ malignancies, including non-small cell lung cancer, triplenegative breast cancer, pancreatic cancer, prostate cancer, ALL, chroniclymphocytic leukemia, or mantle cell lymphoma. For example, MUC16 may betargeted in MUC16ecto+ epithelial ovarian, fallopian tube or primaryperitoneal cancer. For example, CD70 may be targeted in both hematologicmalignancies as well as in solid cancers such as renal cell carcinoma(RCC), gliomas (e.g., GBM), and head and neck cancers (HNSCC). CD70 isexpressed in both hematologic malignancies as well as in solid cancers,while its expression in normal tissues is restricted to a subset oflymphoid cell types (see, e.g., 2018 American Association for CancerResearch (AACR) Annual meeting Poster: Allogeneic CRISPR EngineeredAnti-CD70 CAR-T Cells Demonstrate Potent Preclinical Activity AgainstBoth Solid and Hematological Cancer Cells).

Various strategies may for example be employed to genetically modify Tcells by altering the specificity of the T cell receptor (TCR) forexample by introducing new TCR α and β chains with selected peptidespecificity (see U.S. Pat. No. 8,697,854; PCT Patent Publications:WO2003020763, WO2004033685, WO2004044004, WO2005114215, WO2006000830,WO2008038002, WO2008039818, WO2004074322, WO2005113595, WO2006125962,WO2013166321, WO2013039889, WO2014018863, WO2014083173; U.S. Pat. No.8,088,379).

As an alternative to, or addition to, TCR modifications, chimericantigen receptors (CARs) may be used in order to generateimmunoresponsive cells, such as T cells, specific for selected targets,such as malignant cells, with a wide variety of receptor chimeraconstructs having been described (see U.S. Pat. Nos. 5,843,728;5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014;6,753,162; 8,211,422; and, PCT Publication WO9215322).

In general, CARs are comprised of an extracellular domain, atransmembrane domain, and an intracellular domain, wherein theextracellular domain comprises an antigen-binding domain that isspecific for a predetermined target. While the antigen-binding domain ofa CAR is often an antibody or antibody fragment (e.g., a single chainvariable fragment, scFv), the binding domain is not particularly limitedso long as it results in specific recognition of a target. For example,in some embodiments, the antigen-binding domain may comprise a receptor,such that the CAR is capable of binding to the ligand of the receptor.Alternatively, the antigen-binding domain may comprise a ligand, suchthat the CAR is capable of binding the endogenous receptor of thatligand.

The antigen-binding domain of a CAR is generally separated from thetransmembrane domain by a hinge or spacer. The spacer is also notparticularly limited, and it is designed to provide the CAR withflexibility. For example, a spacer domain may comprise a portion of ahuman Fc domain, including a portion of the CH3 domain, or the hingeregion of any immunoglobulin, such as IgA, IgD, IgE, IgG, or IgM, orvariants thereof. Furthermore, the hinge region may be modified so as toprevent off-target binding by FcRs or other potential interferingobjects. For example, the hinge may comprise an IgG4 Fc domain with orwithout a S228P, L235E, and/or N297Q mutation (according to Kabatnumbering) in order to decrease binding to FcRs. Additionalspacers/hinges include, but are not limited to, CD4, CD8, and CD28 hingeregions.

The transmembrane domain of a CAR may be derived either from a naturalor from a synthetic source. Where the source is natural, the domain maybe derived from any membrane bound or transmembrane protein.Transmembrane regions of particular use in this disclosure may bederived from CD8, CD28, CD3, CD45, CD4, CD5, CDS, CD9, CD 16, CD22,CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, TCR. Alternatively,the transmembrane domain may be synthetic, in which case it willcomprise predominantly hydrophobic residues such as leucine and valine.Preferably a triplet of phenylalanine, tryptophan and valine will befound at each end of a synthetic transmembrane domain. Optionally, ashort oligo- or polypeptide linker, preferably between 2 and 10 aminoacids in length may form the linkage between the transmembrane domainand the cytoplasmic signaling domain of the CAR. A glycine-serinedoublet provides a particularly suitable linker.

Alternative CAR constructs may be characterized as belonging tosuccessive generations. First-generation CARs typically consist of asingle-chain variable fragment of an antibody specific for an antigen,for example comprising a VL linked to a VH of a specific antibody,linked by a flexible linker, for example by a CD8a hinge domain and aCD8a transmembrane domain, to the transmembrane and intracellularsignaling domains of either CD3ζ or FcRγ (scFv-CD3(or scFv-FcRγ; seeU.S. Pat. Nos. 7,741,465; 5,912,172; U.S. Pat. No. 5,906,936).Second-generation CARs incorporate the intracellular domains of one ormore costimulatory molecules, such as CD28, OX40 (CD134), or 4-1BB(CD137) within the endodomain (for example scFv-CD28/OX40/4-1BB-CD3ζ;see U.S. Pat. Nos. 8,911,993; 8,916,381; 8,975,071; 9,101,584;9,102,760; 9,102,761). Third-generation CARs include a combination ofcostimulatory endodomains, such a CD3ζ-chain, CD97, GDI la-CD18, CD2,ICOS, CD27, CD154, CDS, OX40, 4-1BB, CD2, CD7, LIGHT, LFA-1, NKG2C,B7-H3, CD30, CD40, PD-1, or CD28 signaling domains (for examplescFv-CD28-4-1BB-CD3ζ or scFv-CD28—OX40—CD3ζ; see U.S. Pat. Nos.8,906,682; 8,399,645; 5,686,281; PCT Publication No. WO2014134165; PCTPublication No. WO2012079000). In certain embodiments, the primarysignaling domain comprises a functional signaling domain of a proteinselected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta,CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon Rib),CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12. In certain preferredembodiments, the primary signaling domain comprises a functionalsignaling domain of CD3(or FcRγ. In certain embodiments, the one or morecostimulatory signaling domains comprise a functional signaling domainof a protein selected, each independently, from the group consisting of:CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta,IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4,CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a,LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7,TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96(Tactile), CEACAMI, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMFI, CD150, IPO—3),BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44,NKp30, NKp46, and NKG2D. In certain embodiments, the one or morecostimulatory signaling domains comprise a functional signaling domainof a protein selected, each independently, from the group consisting of:4-1BB, CD27, and CD28. In certain embodiments, a chimeric antigenreceptor may have the design as described in U.S. Pat. No. 7,446,190,comprising an intracellular domain of CD3(chain (such as amino acidresidues 52-163 of the human CD3 zeta chain, as shown in SEQ ID NO: 14of U.S. Pat. No. 7,446,190), a signaling region from CD28 and anantigen-binding element (or portion or domain; such as scFv). The CD28portion, when between the zeta chain portion and the antigen-bindingelement, may suitably include the transmembrane and signaling domains ofCD28 (such as amino acid residues 114-220 of SEQ ID NO: 10, fullsequence shown in SEQ ID NO: 6 of U.S. Pat. No. 7,446,190; these caninclude the following portion of CD28 as set forth in Genbank identifierNM_006139 (sequence version 1, 2 or 3):IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS)) (SEQ. I.D. No. 1).Alternatively, when the zeta sequence lies between the CD28 sequence andthe antigen-binding element, intracellular domain of CD28 can be usedalone (such as amino sequence set forth in SEQ ID NO: 9 of U.S. Pat. No.7,446,190). Hence, certain embodiments employ a CAR comprising (a) azeta chain portion comprising the intracellular domain of humanCD3(chain, (b) a costimulatory signaling region, and (c) anantigen-binding element (or portion or domain), wherein thecostimulatory signaling region comprises the amino acid sequence encodedby SEQ ID NO: 6 of U.S. Pat. No. 7,446,190.

Alternatively, costimulation may be orchestrated by expressing CARs inantigen-specific T cells, chosen so as to be activated and expandedfollowing engagement of their native αβTCR, for example by antigen onprofessional antigen-presenting cells, with attendant costimulation. Inaddition, additional engineered receptors may be provided on theimmunoresponsive cells, for example to improve targeting of a T-cellattack and/or minimize side effects

By means of an example and without limitation, Kochenderfer et al.,(2009) J Immunother. 32 (7): 689-702 described anti-CD19 chimericantigen receptors (CAR). FMC63-28Z CAR contained a single chain variableregion moiety (scFv) recognizing CD19 derived from the FMC63 mousehybridoma (described in Nicholson et al., (1997) Molecular Immunology34: 1157-1165), a portion of the human CD28 molecule, and theintracellular component of the human TCR-(molecule. FMC63—CD828BBZ CARcontained the FMC63 scFv, the hinge and transmembrane regions of the CD8molecule, the cytoplasmic portions of CD28 and 4-1BB, and thecytoplasmic component of the TCR-(molecule. The exact sequence of theCD28 molecule included in the FMC63-28Z CAR corresponded to Genbankidentifier NM_006139; the sequence included all amino acids startingwith the amino acid sequence IEVMYPPPY (SEQ. I.D. No. 2) and continuingall the way to the carboxy-terminus of the protein. To encode theanti-CD19 scFv component of the vector, the authors designed a DNAsequence which was based on a portion of a previously published CAR(Cooper et al., (2003) Blood 101: 1637-1644). This sequence encoded thefollowing components in frame from the 5′ end to the 3′ end: an XhoIsite, the human granulocyte-macrophage colony-stimulating factor(GM-CSF) receptor a-chain signal sequence, the FMC63 light chainvariable region (as in Nicholson et al., supra), a linker peptide (as inCooper et al., supra), the FMC63 heavy chain variable region (as inNicholson et al., supra), and a NotI site. A plasmid encoding thissequence was digested with XhoI and NotI. To form the MSGV-FMC63-28Zretroviral vector, the XhoI and NotI-digested fragment encoding theFMC63 scFv was ligated into a second XhoI and NotI-digested fragmentthat encoded the MSGV retroviral backbone (as in Hughes et al., (2005)Human Gene Therapy 16: 457-472) as well as part of the extracellularportion of human CD28, the entire transmembrane and cytoplasmic portionof human CD28, and the cytoplasmic portion of the human TCR-(molecule(as in Maher et al., 2002) Nature Biotechnology 20: 70-75). TheFMC63-28Z CAR is included in the KTE-C19 (axicabtagene ciloleucel)anti-CD19 CAR-T therapy product in development by Kite Pharma, Inc. forthe treatment of inter alia patients with relapsed/refractory aggressiveB-cell non-Hodgkin lymphoma (NHL). Accordingly, in certain embodiments,cells intended for adoptive cell therapies, more particularlyimmunoresponsive cells such as T cells, may express the FMC63-28Z CAR asdescribed by Kochenderfer et al. (supra). Hence, in certain embodiments,cells intended for adoptive cell therapies, more particularlyimmunoresponsive cells such as T cells, may comprise a CAR comprising anextracellular antigen-binding element (or portion or domain; such asscFv) that specifically binds to an antigen, an intracellular signalingdomain comprising an intracellular domain of a CD3(chain, and acostimulatory signaling region comprising a signaling domain of CD28.Preferably, the CD28 amino acid sequence is as set forth in Genbankidentifier NM_006139 (sequence version 1, 2 or 3) starting with theamino acid sequence IEVMYPPPY and continuing all the way to thecarboxy-terminus of the protein. The sequence is reproduced herein:IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS. Preferably, the antigenis CD19, more preferably the antigen-binding element is an anti-CD19scFv, even more preferably the anti-CD19 scFv as described byKochenderfer et al. (supra).

Additional anti-CD19 CARs are further described in WO2015187528. Moreparticularly Example 1 and Table 1 of WO2015187528, incorporated byreference herein, demonstrate the generation of anti-CD19 CARs based ona fully human anti-CD19 monoclonal antibody (47G4, as described inUS20100104509) and murine anti-CD19 monoclonal antibody (as described inNicholson et al. and explained above). Various combinations of a signalsequence (human CD8-alpha or GM-CSF receptor), extracellular andtransmembrane regions (human CD8-alpha) and intracellular T-cellsignalling domains (CD28—CD3ζ; 4-1BB-CD3ζ; CD27—CD3ζ; CD28—CD27—CD3ζ,4-1BB-CD27—CD3ζ; CD27-4-1BB-CD3ζ; CD28—CD27-FcgRI gamma chain; orCD28-FcεFRI gamma chain) were disclosed. Hence, in certain embodiments,cells intended for adoptive cell therapies, more particularlyimmunoresponsive cells such as T cells, may comprise a CAR comprising anextracellular antigen-binding element that specifically binds to anantigen, an extracellular and transmembrane region as set forth in Table1 of WO2015187528 and an intracellular T-cell signalling domain as setforth in Table 1 of WO2015187528. Preferably, the antigen is CD19, morepreferably the antigen-binding element is an anti-CD19 scFv, even morepreferably the mouse or human anti-CD19 scFv as described in Example 1of WO2015187528. In certain embodiments, the CAR comprises, consistsessentially of or consists of an amino acid sequence of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, or SEQ ID NO: 13 as set forth in Table 1 of WO2015187528.

By means of an example and without limitation, chimeric antigen receptorthat recognizes the CD70 antigen is described in WO2012058460A2 (seealso, Park et al., CD70 as a target for chimeric antigen receptor Tcells in head and neck squamous cell carcinoma, Oral Oncol. 2018March;78:145-150; and Jin et al., CD70, a novel target of CAR T-celltherapy for gliomas, Neuro Oncol. 2018 Jan. 10; 20(1):55-65). CD70 isexpressed by diffuse large B-cell and follicular lymphoma and also bythe malignant cells of Hodgkins lymphoma, Waldenstrom'smacroglobulinemia and multiple myeloma, and by HTLV-1- andEBV-associated malignancies. (Agathanggelou et al. Am.J.Pathol. 1995;147: 1152-1160; Hunter et al., Blood 2004; 104:4881. 26; Lens et al., JImmunol. 2005; 174:6212-6219; Baba et al., J Virol. 2008; 82:3843-3852.)In addition, CD70 is expressed by non-hematological malignancies such asrenal cell carcinoma and glioblastoma. (Junker et al., J Urol. 2005;173:2150-2153; Chahlavi et al., Cancer Res 2005; 65:5428-5438)Physiologically, CD70 expression is transient and restricted to a subsetof highly activated T, B, and dendritic cells.

By means of an example and without limitation, chimeric antigen receptorthat recognizes BCMA has been described (see, e.g., US20160046724A1;WO2016014789A2; WO2017211900A1; WO2015158671A1; US20180085444A1;WO2018028647A1; US20170283504A1; and WO2013154760A1).

In certain embodiments, the immune cell may, in addition to a CAR orexogenous TCR as described herein, further comprise a chimericinhibitory receptor (inhibitory CAR) that specifically binds to a secondtarget antigen and is capable of inducing an inhibitory orimmunosuppressive or repressive signal to the cell upon recognition ofthe second target antigen. In certain embodiments, the chimericinhibitory receptor comprises an extracellular antigen-binding element(or portion or domain) configured to specifically bind to a targetantigen, a transmembrane domain, and an intracellular immunosuppressiveor repressive signaling domain. In certain embodiments, the secondtarget antigen is an antigen that is not expressed on the surface of acancer cell or infected cell or the expression of which is downregulatedon a cancer cell or an infected cell. In certain embodiments, the secondtarget antigen is an MHC-class I molecule. In certain embodiments, theintracellular signaling domain comprises a functional signaling portionof an immune checkpoint molecule, such as for example PD-1 or CTLA4.Advantageously, the inclusion of such inhibitory CAR reduces the chanceof the engineered immune cells attacking non-target (e.g., non-cancer)tissues.

Alternatively, T-cells expressing CARs may be further modified to reduceor eliminate expression of endogenous TCRs in order to reduce off-targeteffects. Reduction or elimination of endogenous TCRs can reduceoff-target effects and increase the effectiveness of the T cells (U.S.Pat. No. 9,181,527). T cells stably lacking expression of a functionalTCR may be produced using a variety of approaches. T cells internalize,sort, and degrade the entire T cell receptor as a complex, with ahalf-life of about 10 hours in resting T cells and 3 hours in stimulatedT cells (von Essen, M. et al. 2004. J. Immunol. 173:384-393). Properfunctioning of the TCR complex requires the proper stoichiometric ratioof the proteins that compose the TCR complex. TCR function also requirestwo functioning TCR zeta proteins with ITAM motifs. The activation ofthe TCR upon engagement of its MHC-peptide ligand requires theengagement of several TCRs on the same T cell, which all must signalproperly. Thus, if a TCR complex is destabilized with proteins that donot associate properly or cannot signal optimally, the T cell will notbecome activated sufficiently to begin a cellular response.

Accordingly, in some embodiments, TCR expression may eliminated usingRNA interference (e.g., shRNA, siRNA, miRNA, etc.), CRISPR, or othermethods that target the nucleic acids encoding specific TCRs (e.g.,TCR-α and TCR-β) and/or CD3 chains in primary T cells. By blockingexpression of one or more of these proteins, the T cell will no longerproduce one or more of the key components of the TCR complex, therebydestabilizing the TCR complex and preventing cell surface expression ofa functional TCR.

In some instances, CAR may also comprise a switch mechanism forcontrolling expression and/or activation of the CAR. For example, a CARmay comprise an extracellular, transmembrane, and intracellular domain,in which the extracellular domain comprises a target-specific bindingelement that comprises a label, binding domain, or tag that is specificfor a molecule other than the target antigen that is expressed on or bya target cell. In such embodiments, the specificity of the CAR isprovided by a second construct that comprises a target antigen bindingdomain (e.g., an scFv or a bispecific antibody that is specific for boththe target antigen and the label or tag on the CAR) and a domain that isrecognized by or binds to the label, binding domain, or tag on the CAR.See, e.g., WO 2013/044225, WO 2016/000304, WO 2015/057834, WO2015/057852, WO 2016/070061, U.S. Pat. No. 9,233,125, US 2016/0129109.In this way, a T-cell that expresses the CAR can be administered to asubject, but the CAR cannot bind its target antigen until the secondcomposition comprising an antigen-specific binding domain isadministered.

Alternative switch mechanisms include CARs that require multimerizationin order to activate their signaling function (see, e.g., US2015/0368342, US 2016/0175359, US 2015/0368360) and/or an exogenoussignal, such as a small molecule drug (US 2016/0166613, Yung et al.,Science, 2015), in order to elicit a T-cell response. Some CARs may alsocomprise a “suicide switch” to induce cell death of the CAR T-cellsfollowing treatment (Buddee et al., PLoS One, 2013) or to downregulateexpression of the CAR following binding to the target antigen (WO2016/011210).

Alternative techniques may be used to transform target immunoresponsivecells, such as protoplast fusion, lipofection, transfection orelectroporation. A wide variety of vectors may be used, such asretroviral vectors, lentiviral vectors, adenoviral vectors,adeno-associated viral vectors, plasmids or transposons, such as aSleeping Beauty transposon (see U.S. Pat. Nos. 6,489,458; 7,148,203;7,160,682; 7,985,739; 8,227,432), may be used to introduce CARs, forexample using 2nd generation antigen-specific CARs signaling throughCD3(and either CD28 or CD137. Viral vectors may for example includevectors based on HIV, SV40, EBV, HSV or BPV.

Cells that are targeted for transformation may for example include Tcells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL),regulatory T cells, human embryonic stem cells, tumor-infiltratinglymphocytes (TIL) or a pluripotent stem cell from which lymphoid cellsmay be differentiated. T cells expressing a desired CAR may for examplebe selected through co-culture with y-irradiated activating andpropagating cells (AaPC), which co-express the cancer antigen andco-stimulatory molecules. The engineered CAR T-cells may be expanded,for example by co-culture on AaPC in presence of soluble factors, suchas IL-2 and IL-21. This expansion may for example be carried out so asto provide memory CAR+ T cells (which may for example be assayed bynon-enzymatic digital array and/or multi-panel flow cytometry). In thisway, CAR T cells may be provided that have specific cytotoxic activityagainst antigen-bearing tumors (optionally in conjunction withproduction of desired chemokines such as interferon-y). CAR T cells ofthis kind may for example be used in animal models, for example to treattumor xenografts.

In certain embodiments, ACT includes co-transferring CD4+ Th1 cells andCD8+ CTLs to induce a synergistic antitumour response (see, e.g., Li etal., Adoptive cell therapy with CD4+ T helper 1 cells and CD8+ cytotoxicT cells enhances complete rejection of an established tumour, leading togeneration of endogenous memory responses to non-targeted tumourepitopes. Clin Transl Immunology. 2017 October; 6(10): e160).

In certain embodiments, Th17 cells are transferred to a subject in needthereof Th17 cells have been reported to directly eradicate melanomatumors in mice to a greater extent than Th1 cells (Muranski P, et al.,Tumor-specific Th17-polarized cells eradicate large establishedmelanoma. Blood. 2008 Jul. 15; 112(2):362-73; and Martin-Orozco N, etal., T helper 17 cells promote cytotoxic T cell activation in tumorimmunity. Immunity. 2009 Nov. 20; 31(5):787-98). Those studies involvedan adoptive T cell transfer (ACT) therapy approach, which takesadvantage of CD4+ T cells that express a TCR recognizing tyrosinasetumor antigen. Exploitation of the TCR leads to rapid expansion of Th17populations to large numbers ex vivo for reinfusion into the autologoustumor-bearing hosts.

In certain embodiments, ACT may include autologous iPSC-based vaccines,such as irradiated iPSCs in autologous anti-tumor vaccines (see e.g.,Kooreman, Nigel G. et al., Autologous iPSC-Based Vaccines ElicitAnti-tumor Responses In Vivo, Cell Stem Cell 22, 1-13, 2018,doi.org/10.1016/j.stem.2018.01.016).

Unlike T-cell receptors (TCRs) that are MHC restricted, CARs canpotentially bind any cell surface-expressed antigen and can thus be moreuniversally used to treat patients (see Irving et al., EngineeringChimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don'tForget the Fuel, Front. Immunol., 3 Apr. 2017,doi.org/10.3389/fimmu.2017.00267). In certain embodiments, in theabsence of endogenous T-cell infiltrate (e.g., due to aberrant antigenprocessing and presentation), which precludes the use of TIL therapy andimmune checkpoint blockade, the transfer of CAR T-cells may be used totreat patients (see, e.g., Hinrichs C S, Rosenberg S A. Exploiting thecurative potential of adoptive T-cell therapy for cancer. Immunol Rev(2014) 257(1):56-71. doi:10.1111/imr.12132).

Approaches such as the foregoing may be adapted to provide methods oftreating and/or increasing survival of a subject having a disease, suchas a neoplasia, for example by administering an effective amount of animmunoresponsive cell comprising an antigen recognizing receptor thatbinds a selected antigen, wherein the binding activates theimmunoresponsive cell, thereby treating or preventing the disease (suchas a neoplasia, a pathogen infection, an autoimmune disorder, or anallogeneic transplant reaction).

In certain embodiments, the treatment can be administered afterlymphodepleting pretreatment in the form of chemotherapy (typically acombination of cyclophosphamide and fludarabine) or radiation therapy.Initial studies in ACT had short lived responses and the transferredcells did not persist in vivo for very long (Houot et al., T-cell-basedimmunotherapy: adoptive cell transfer and checkpoint inhibition. CancerImmunol Res (2015) 3(10):1115-22; and Kamta et al., Advancing CancerTherapy with Present and Emerging Immuno-Oncology Approaches. Front.Oncol. (2017) 7:64). Immune suppressor cells like Tregs and MDSCs mayattenuate the activity of transferred cells by outcompeting them for thenecessary cytokines. Not being bound by a theory lymphodepletingpretreatment may eliminate the suppressor cells allowing the TILs topersist.

In one embodiment, the treatment can be administrated into patientsundergoing an immunosuppressive treatment (e.g., glucocorticoidtreatment). The cells or population of cells, may be made resistant toat least one immunosuppressive agent due to the inactivation of a geneencoding a receptor for such immunosuppressive agent. In certainembodiments, the immunosuppressive treatment provides for the selectionand expansion of the immunoresponsive T cells within the patient.

In certain embodiments, the treatment can be administered before primarytreatment (e.g., surgery or radiation therapy) to shrink a tumor beforethe primary treatment. In another embodiment, the treatment can beadministered after primary treatment to remove any remaining cancercells.

In certain embodiments, immunometabolic barriers can be targetedtherapeutically prior to and/or during ACT to enhance responses to ACTor CAR T-cell therapy and to support endogenous immunity (see, e.g.,Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racingin Solid Tumors: Don't Forget the Fuel, Front. Immunol., 3 Apr. 2017,doi.org/10.3389/fimmu.2017.00267).

The administration of cells or population of cells, such as immunesystem cells or cell populations, such as more particularlyimmunoresponsive cells or cell populations, as disclosed herein may becarried out in any convenient manner, including by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation. Thecells or population of cells may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, intrathecally, by intravenous orintralymphatic injection, or intraperitoneally. In some embodiments, thedisclosed CARs may be delivered or administered into a cavity formed bythe resection of tumor tissue (i.e. intracavity delivery) or directlyinto a tumor prior to resection (i.e. intratumoral delivery). In oneembodiment, the cell compositions of the present invention arepreferably administered by intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 104-109 cells per kg body weight, preferably 10¹to 10⁶ cells/kg body weight including all integer values of cell numberswithin those ranges. Dosing in CAR T cell therapies may for exampleinvolve administration of from 10⁶ to 109 cells/kg, with or without acourse of lymphodepletion, for example with cyclophosphamide. The cellsor population of cells can be administrated in one or more doses. Inanother embodiment, the effective amount of cells are administrated as asingle dose. In another embodiment, the effective amount of cells areadministrated as more than one dose over a period time. Timing ofadministration is within the judgment of managing physician and dependson the clinical condition of the patient. The cells or population ofcells may be obtained from any source, such as a blood bank or a donor.While individual needs vary, determination of optimal ranges ofeffective amounts of a given cell type for a particular disease orconditions are within the skill of one in the art. An effective amountmeans an amount which provides a therapeutic or prophylactic benefit.The dosage administrated will be dependent upon the age, health andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment and the nature of the effect desired.

In another embodiment, the effective amount of cells or compositioncomprising those cells are administrated parenterally. Theadministration can be an intravenous administration. The administrationcan be directly done by injection within a tumor.

To guard against possible adverse reactions, engineered immunoresponsivecells may be equipped with a transgenic safety switch, in the form of atransgene that renders the cells vulnerable to exposure to a specificsignal. For example, the herpes simplex viral thymidine kinase (TK) genemay be used in this way, for example by introduction into allogeneic Tlymphocytes used as donor lymphocyte infusions following stem celltransplantation (Greco, et al., Improving the safety of cell therapywith the TK-suicide gene. Front. Pharmacol. 2015; 6: 95). In such cells,administration of a nucleoside prodrug such as ganciclovir or acyclovircauses cell death. Alternative safety switch constructs includeinducible caspase 9, for example triggered by administration of asmall-molecule dimerizer that brings together two nonfunctional icasp9molecules to form the active enzyme. A wide variety of alternativeapproaches to implementing cellular proliferation controls have beendescribed (see U.S. Patent Publication No. 20130071414; PCT PatentPublication WO2011146862; PCT Patent Publication WO2014011987; PCTPatent Publication WO2013040371; Zhou et al. BLOOD, 2014,123/25:3895-3905; Di Stasi et al., The New England Journal of Medicine2011; 365:1673-1683; Sadelain M, The New England Journal of Medicine2011; 365:1735-173; Ramos et al., Stem Cells 28(6):1107-15 (2010)).

In a further refinement of adoptive therapies, genome editing may beused to tailor immunoresponsive cells to alternative implementations,for example providing edited CAR T cells (see Poirot et al., 2015,Multiplex genome edited T-cell manufacturing platform for“off-the-shelf” adoptive T-cell immunotherapies, Cancer Res 75 (18):3853; Ren et al., 2017, Multiplex genome editing to generate universalCAR T cells resistant to PD1 inhibition, Clin Cancer Res. 2017 May 1;23(9):2255-2266. doi: 10.1158/1078-0432.CCR-16-1300. Epub 2016 Nov. 4;Qasim et al., 2017, Molecular remission of infant B-ALL after infusionof universal TALEN gene-edited CAR T cells, Sci Transl Med. 2017 Jan.25; 9(374); Legut, et al., 2018, CRISPR-mediated TCR replacementgenerates superior anticancer transgenic T cells. Blood, 131(3),311-322; and Georgiadis et al., Long Terminal Repeat CRISPR-CAR-Coupled“Universal” T Cells Mediate Potent Anti-leukemic Effects, MolecularTherapy, In Press, Corrected Proof, Available online 6 Mar. 2018). Cellsmay be edited using any CRISPR system and method of use thereof asdescribed herein. CRISPR systems may be delivered to an immune cell byany method described herein. In preferred embodiments, cells are editedex vivo and transferred to a subject in need thereof. Immunoresponsivecells, CAR T cells or any cells used for adoptive cell transfer may beedited. Editing may be performed for example to insert or knock-in anexogenous gene, such as an exogenous gene encoding a CAR or a TCR, at apreselected locus in a cell (e.g. TRAC locus); to eliminate potentialalloreactive T-cell receptors (TCR) or to prevent inappropriate pairingbetween endogenous and exogenous TCR chains, such as to knock-out orknock-down expression of an endogenous TCR in a cell; to disrupt thetarget of a chemotherapeutic agent in a cell; to block an immunecheckpoint, such as to knock-out or knock-down expression of an immunecheckpoint protein or receptor in a cell; to knock-out or knock-downexpression of other gene or genes in a cell, the reduced expression orlack of expression of which can enhance the efficacy of adoptivetherapies using the cell; to knock-out or knock-down expression of anendogenous gene in a cell, said endogenous gene encoding an antigentargeted by an exogenous CAR or TCR; to knock-out or knock-downexpression of one or more MHC constituent proteins in a cell; toactivate a T cell; to modulate cells such that the cells are resistantto exhaustion or dysfunction; and/or increase the differentiation and/orproliferation of functionally exhausted or dysfunctional CD8+ T-cells(see PCT Patent Publications: WO2013176915, WO2014059173, WO2014172606,WO2014184744, and WO2014191128).

In certain embodiments, editing may result in inactivation of a gene. Byinactivating a gene, it is intended that the gene of interest is notexpressed in a functional protein form. In a particular embodiment, theCRISPR system specifically catalyzes cleavage in one targeted genethereby inactivating said targeted gene. The nucleic acid strand breakscaused are commonly repaired through the distinct mechanisms ofhomologous recombination or non-homologous end joining (NHEJ). However,NHEJ is an imperfect repair process that often results in changes to theDNA sequence at the site of the cleavage. Repair via non-homologous endjoining (NHEJ) often results in small insertions or deletions (Indel)and can be used for the creation of specific gene knockouts. Cells inwhich a cleavage induced mutagenesis event has occurred can beidentified and/or selected by well-known methods in the art. In certainembodiments, homology directed repair (HDR) is used to concurrentlyinactivate a gene (e.g., TRAC) and insert an endogenous TCR or CAR intothe inactivated locus.

Hence, in certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toinsert or knock-in an exogenous gene, such as an exogenous gene encodinga CAR or a TCR, at a preselected locus in a cell. Conventionally,nucleic acid molecules encoding CARs or TCRs are transfected ortransduced to cells using randomly integrating vectors, which, dependingon the site of integration, may lead to clonal expansion, oncogenictransformation, variegated transgene expression and/or transcriptionalsilencing of the transgene. Directing of transgene(s) to a specificlocus in a cell can minimize or avoid such risks and advantageouslyprovide for uniform expression of the transgene(s) by the cells. Withoutlimitation, suitable ‘safe harbor’ loci for directed transgeneintegration include CCR5 or AAVS1. Homology-directed repair (HDR)strategies are known and described elsewhere in this specificationallowing to insert transgenes into desired loci (e.g., TRAC locus).

Further suitable loci for insertion of transgenes, in particular CAR orexogenous TCR transgenes, include without limitation loci comprisinggenes coding for constituents of endogenous T-cell receptor, such asT-cell receptor alpha locus (TRA) or T-cell receptor beta locus (TRB),for example T-cell receptor alpha constant (TRAC) locus, T-cell receptorbeta constant 1 (TRBC1) locus or T-cell receptor beta constant 2 (TRBC1)locus. Advantageously, insertion of a transgene into such locus cansimultaneously achieve expression of the transgene, potentiallycontrolled by the endogenous promoter, and knock-out expression of theendogenous TCR. This approach has been exemplified in Eyquem et al.,(2017) Nature 543: 113-117, wherein the authors used CRISPR/Cas9 geneediting to knock-in a DNA molecule encoding a CD19-specific CAR into theTRAC locus downstream of the endogenous promoter; the CAR-T cellsobtained by CRISPR were significantly superior in terms of reduced tonicCAR signaling and exhaustion.

T cell receptors (TCR) are cell surface receptors that participate inthe activation of T cells in response to the presentation of antigen.The TCR is generally made from two chains, α and β, which assemble toform a heterodimer and associates with the CD3-transducing subunits toform the T cell receptor complex present on the cell surface. Each a andp chain of the TCR consists of an immunoglobulin-like N-terminalvariable (V) and constant (C) region, a hydrophobic transmembranedomain, and a short cytoplasmic region. As for immunoglobulin molecules,the variable region of the a and p chains are generated by V(D)Jrecombination, creating a large diversity of antigen specificitieswithin the population of T cells. However, in contrast toimmunoglobulins that recognize intact antigen, T cells are activated byprocessed peptide fragments in association with an MHC molecule,introducing an extra dimension to antigen recognition by T cells, knownas MHC restriction. Recognition of MHC disparities between the donor andrecipient through the T cell receptor leads to T cell proliferation andthe potential development of graft versus host disease (GVHD). Theinactivation of TCRα or TCRβ can result in the elimination of the TCRfrom the surface of T cells preventing recognition of alloantigen andthus GVHD. However, TCR disruption generally results in the eliminationof the CD3 signaling component and alters the means of further T cellexpansion.

Hence, in certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toknock-out or knock-down expression of an endogenous TCR in a cell. Forexample, NHEJ-based or HDR-based gene editing approaches can be employedto disrupt the endogenous TCR alpha and/or beta chain genes. Forexample, gene editing system or systems, such as CRISPR/Cas system orsystems, can be designed to target a sequence found within the TCR betachain conserved between the beta 1 and beta 2 constant region genes(TRBC1 and TRBC2) and/or to target the constant region of the TCR alphachain (TRAC) gene.

Allogeneic cells are rapidly rejected by the host immune system. It hasbeen demonstrated that, allogeneic leukocytes present in non-irradiatedblood products will persist for no more than 5 to 6 days (Boni, Muranskiet al. 2008 Blood 1; 112(12):4746-54). Thus, to prevent rejection ofallogeneic cells, the host's immune system usually has to be suppressedto some extent. However, in the case of adoptive cell transfer the useof immunosuppressive drugs also have a detrimental effect on theintroduced therapeutic T cells. Therefore, to effectively use anadoptive immunotherapy approach in these conditions, the introducedcells would need to be resistant to the immunosuppressive treatment.Thus, in a particular embodiment, the present invention furthercomprises a step of modifying T cells to make them resistant to animmunosuppressive agent, preferably by inactivating at least one geneencoding a target for an immunosuppressive agent. An immunosuppressiveagent is an agent that suppresses immune function by one of severalmechanisms of action. An immunosuppressive agent can be, but is notlimited to a calcineurin inhibitor, a target of rapamycin, aninterleukin-2 receptor a-chain blocker, an inhibitor of inosinemonophosphate dehydrogenase, an inhibitor of dihydrofolic acidreductase, a corticosteroid or an immunosuppressive antimetabolite. Thepresent invention allows conferring immunosuppressive resistance to Tcells for immunotherapy by inactivating the target of theimmunosuppressive agent in T cells. As non-limiting examples, targetsfor an immunosuppressive agent can be a receptor for animmunosuppressive agent such as: CD52, glucocorticoid receptor (GR), aFKBP family gene member and a cyclophilin family gene member.

In certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toblock an immune checkpoint, such as to knock-out or knock-downexpression of an immune checkpoint protein or receptor in a cell. Immunecheckpoints are inhibitory pathways that slow down or stop immunereactions and prevent excessive tissue damage from uncontrolled activityof immune cells. In certain embodiments, the immune checkpoint targetedis the programmed death-1 (PD-1 or CD279) gene (PDCD1). In otherembodiments, the immune checkpoint targeted is cytotoxicT-lymphocyte-associated antigen (CTLA-4). In additional embodiments, theimmune checkpoint targeted is another member of the CD28 and CTLA4 Igsuperfamily such as BTLA, LAG3, ICOS, PDL1 or KIR. In further additionalembodiments, the immune checkpoint targeted is a member of the TNFRsuperfamily such as CD40, OX40, CD137, GITR, CD27 or TIM-3.

Additional immune checkpoints include Src homology 2 domain-containingprotein tyrosine phosphatase 1 (SHP-1) (Watson H A, et al., SHP-1: thenext checkpoint target for cancer immunotherapy? Biochem Soc Trans. 2016Apr. 2015; 44(2):356-62). SHP-1 is a widely expressed inhibitory proteintyrosine phosphatase (PTP). In T-cells, it is a negative regulator ofantigen-dependent activation and proliferation. It is a cytosolicprotein, and therefore not amenable to antibody-mediated therapies, butits role in activation and proliferation makes it an attractive targetfor genetic manipulation in adoptive transfer strategies, such aschimeric antigen receptor (CAR) T cells. Immune checkpoints may alsoinclude T cell immunoreceptor with Ig and ITIM domains(TIGIT/Vstm3/WUCAM/VSIG9) and VISTA (Le Mercier I, et al., (2015) BeyondCTLA-4 and PD-1, the generation Z of negative checkpoint regulators.Front. Immunol. 6:418).

WO2014172606 relates to the use of MT1 and/or MT2 inhibitors to increaseproliferation and/or activity of exhausted CD8+ T-cells and to decreaseCD8+ T-cell exhaustion (e.g., decrease functionally exhausted orunresponsive CD8+ immune cells). In certain embodiments,metallothioneins are targeted by gene editing in adoptively transferredT cells.

In certain embodiments, targets of gene editing may be at least onetargeted locus involved in the expression of an immune checkpointprotein. Such targets may include, but are not limited to CTLA4, PPP2CA,PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2,BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4),TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS,TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA,IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1,BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, MT1, MT2, CD40, OX40,CD137, GITR, CD27, SHP-1, TIM-3, CEACAM-1, CEACAM-3, or CEACAM-5. Inpreferred embodiments, the gene locus involved in the expression of PD-1or CTLA-4 genes is targeted. In other preferred embodiments,combinations of genes are targeted, such as but not limited to PD-1 andTIGIT.

By means of an example and without limitation, WO2016196388 concerns anengineered T cell comprising (a) a genetically engineered antigenreceptor that specifically binds to an antigen, which receptor may be aCAR; and (b) a disrupted gene encoding a PD-L1, an agent for disruptionof a gene encoding a PD- LI, and/or disruption of a gene encoding PD-LI,wherein the disruption of the gene may be mediated by a gene editingnuclease, a zinc finger nuclease (ZFN), CRISPR/Cas9 and/or TALEN.WO2015142675 relates to immune effector cells comprising a CAR incombination with an agent (such as CRISPR, TALEN or ZFN) that increasesthe efficacy of the immune effector cells in the treatment of cancer,wherein the agent may inhibit an immune inhibitory molecule, such asPD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4,TGFR beta, CEACAM-1, CEACAM-3, or CEACAM-5. Ren et al., (2017) ClinCancer Res 23 (9) 2255-2266 performed lentiviral delivery of CAR andelectro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, 3-2microglobulin (B2M) and PD1 simultaneously, to generate gene-disruptedallogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.

In certain embodiments, cells may be engineered to express a CAR,wherein expression and/or function of methylcytosine dioxygenase genes(TET1, TET2 and/or TET3) in the cells has been reduced or eliminated,such as by CRISPR, ZNF or TALEN (for example, as described inWO201704916).

In certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toknock-out or knock-down expression of an endogenous gene in a cell, saidendogenous gene encoding an antigen targeted by an exogenous CAR or TCR,thereby reducing the likelihood of targeting of the engineered cells. Incertain embodiments, the targeted antigen may be one or more antigenselected from the group consisting of CD38, CD138, CS-1, CD33, CD26,CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, CD362, humantelomerase reverse transcriptase (hTERT), survivin, mouse double minute2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumorgene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen(CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen(PSMA), p53, cyclin (D1), B cell maturation antigen (BCMA),transmembrane activator and CAML Interactor (TACI), and B-cellactivating factor receptor (BAFF-R) (for example, as described inWO2016011210 and WO2017011804).

In certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toknock-out or knock-down expression of one or more MHC constituentproteins, such as one or more HLA proteins and/or beta-2 microglobulin(B2M), in a cell, whereby rejection of non-autologous (e.g., allogeneic)cells by the recipient's immune system can be reduced or avoided. Inpreferred embodiments, one or more HLA class I proteins, such as HLA-A,B and/or C, and/or B2M may be knocked-out or knocked-down. Preferably,B2M may be knocked-out or knocked-down. By means of an example, Ren etal., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviraldelivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targetingendogenous TCR, J-2 microglobulin (B2M) and PD1 simultaneously, togenerate gene-disrupted allogeneic CAR T cells deficient of TCR, HLAclass I molecule and PD1.

In other embodiments, at least two genes are edited. Pairs of genes mayinclude, but are not limited to PD1 and TCRα, PD1 and TCRβ, CTLA-4 andTCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ,TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 andTCRα, LAIR1 and TCRβ, SIGLEC1O and TCRα, SIGLEC1O and TCRβ, 2B4 andTCRα, 2B4 and TCRβ, B2M and TCRα, B2M and TCRβ .

In certain embodiments, a cell may be multiply edited (multiplex genomeediting) as taught herein to (1) knock-out or knock-down expression ofan endogenous TCR (for example, TRBC1, TRBC2 and/or TRAC), (2) knock-outor knock-down expression of an immune checkpoint protein or receptor(for example PD1, PD-L1 and/or CTLA4); and (3) knock-out or knock-downexpression of one or more MHC constituent proteins (for example, HLA-A,B and/or C, and/or B2M, preferably B2M).

Whether prior to or after genetic modification of the T cells, the Tcells can be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566;7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631. Tcells can be expanded in vitro or in vivo.

Immune cells may be obtained using any method known in the art. In oneembodiment, allogenic T cells may be obtained from healthy subjects. Inone embodiment T cells that have infiltrated a tumor are isolated. Tcells may be removed during surgery. T cells may be isolated afterremoval of tumor tissue by biopsy. T cells may be isolated by any meansknown in the art. In one embodiment, T cells are obtained by apheresis.In one embodiment, the method may comprise obtaining a bulk populationof T cells from a tumor sample by any suitable method known in the art.For example, a bulk population of T cells can be obtained from a tumorsample by dissociating the tumor sample into a cell suspension fromwhich specific cell populations can be selected. Suitable methods ofobtaining a bulk population of T cells may include, but are not limitedto, any one or more of mechanically dissociating (e.g., mincing) thetumor, enzymatically dissociating (e.g., digesting) the tumor, andaspiration (e.g., as with a needle).

The bulk population of T cells obtained from a tumor sample may compriseany suitable type of T cell. Preferably, the bulk population of T cellsobtained from a tumor sample comprises tumor infiltrating lymphocytes(TILs).

The tumor sample may be obtained from any mammal. Unless statedotherwise, as used herein, the term “mammal” refers to any mammalincluding, but not limited to, mammals of the order Logomorpha, such asrabbits; the order Carnivora, including Felines (cats) and Canines(dogs); the order Artiodactyla, including Bovines (cows) and Swines(pigs); or of the order Perssodactyla, including Equines (horses). Themammals may be non-human primates, e.g., of the order Primates, Ceboids,or Simoids (monkeys) or of the order Anthropoids (humans and apes). Insome embodiments, the mammal may be a mammal of the order Rodentia, suchas mice and hamsters. Preferably, the mammal is a non-human primate or ahuman. An especially preferred mammal is the human.

T cells can be obtained from a number of sources, including peripheralblood mononuclear cells (PBMC), bone marrow, lymph node tissue, spleentissue, and tumors. In certain embodiments of the present invention, Tcells can be obtained from a unit of blood collected from a subjectusing any number of techniques known to the skilled artisan, such asFicoll separation. In one preferred embodiment, cells from thecirculating blood of an individual are obtained by apheresis orleukapheresis. The apheresis product typically contains lymphocytes,including T cells, monocytes, granulocytes, B cells, other nucleatedwhite blood cells, red blood cells, and platelets. In one embodiment,the cells collected by apheresis may be washed to remove the plasmafraction and to place the cells in an appropriate buffer or media forsubsequent processing steps. In one embodiment of the invention, thecells are washed with phosphate buffered saline (PBS). In an alternativeembodiment, the wash solution lacks calcium and may lack magnesium ormay lack many if not all divalent cations. Initial activation steps inthe absence of calcium lead to magnified activation. As those ofordinary skill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor) according to the manufacturer's instructions. Afterwashing, the cells may be resuspended in a variety of biocompatiblebuffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theundesirable components of the apheresis sample may be removed and thecells directly resuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient. A specificsubpopulation of T cells, such as CD28+, CD4+, CDC, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selectiontechniques. For example, in one preferred embodiment, T cells areisolated by incubation with anti-CD3/anti-CD28 (i.e., 3×28)-conjugatedbeads, such as DYNABEADS® M-450 CD3/CD28 T, or XCYTE DYNABEADS™ for atime period sufficient for positive selection of the desired T cells. Inone embodiment, the time period is about 30 minutes. In a furtherembodiment, the time period ranges from 30 minutes to 36 hours or longerand all integer values there between. In a further embodiment, the timeperiod is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferredembodiment, the time period is 10 to 24 hours. In one preferredembodiment, the incubation time period is 24 hours. For isolation of Tcells from patients with leukemia, use of longer incubation times, suchas 24 hours, can increase cell yield. Longer incubation times may beused to isolate T cells in any situation where there are few T cells ascompared to other cell types, such in isolating tumor infiltratinglymphocytes (TIL) from tumor tissue or from immunocompromisedindividuals. Further, use of longer incubation times can increase theefficiency of capture of CD8+ T cells.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. A preferred method iscell sorting and/or selection via negative magnetic immunoadherence orflow cytometry that uses a cocktail of monoclonal antibodies directed tocell surface markers present on the cells negatively selected. Forexample, to enrich for CD4+ cells by negative selection, a monoclonalantibody cocktail typically includes antibodies to CD14, CD20, CD11b,CD16, HLA-DR, and CD8.

Further, monocyte populations (i.e., CD14+ cells) may be depleted fromblood preparations by a variety of methodologies, including anti-CD14coated beads or columns, or utilization of the phagocytotic activity ofthese cells to facilitate removal. Accordingly, in one embodiment, theinvention uses paramagnetic particles of a size sufficient to beengulfed by phagocytotic monocytes. In certain embodiments, theparamagnetic particles are commercially available beads, for example,those produced by Life Technologies under the trade name Dynabeads™. Inone embodiment, other non-specific cells are removed by coating theparamagnetic particles with “irrelevant” proteins (e.g., serum proteinsor antibodies). Irrelevant proteins and antibodies include thoseproteins and antibodies or fragments thereof that do not specificallytarget the T cells to be isolated. In certain embodiments, theirrelevant beads include beads coated with sheep anti-mouse antibodies,goat anti-mouse antibodies, and human serum albumin.

In brief, such depletion of monocytes is performed by preincubating Tcells isolated from whole blood, apheresed peripheral blood, or tumorswith one or more varieties of irrelevant or non-antibody coupledparamagnetic particles at any amount that allows for removal ofmonocytes (approximately a 20:1 bead:cell ratio) for about 30 minutes to2 hours at 22 to 37 degrees C., followed by magnetic removal of cellswhich have attached to or engulfed the paramagnetic particles. Suchseparation can be performed using standard methods available in the art.For example, any magnetic separation methodology may be used including avariety of which are commercially available, (e.g., DYNAL® MagneticParticle Concentrator (DYNAL MPC®)). Assurance of requisite depletioncan be monitored by a variety of methodologies known to those ofordinary skill in the art, including flow cytometric analysis of CD14positive cells, before and after depletion.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8+ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

T cells can also be frozen. Wishing not to be bound by theory, thefreeze and subsequent thaw step provides a more uniform product byremoving granulocytes and to some extent monocytes in the cellpopulation. After a washing step to remove plasma and platelets, thecells may be suspended in a freezing solution. While many freezingsolutions and parameters are known in the art and will be useful in thiscontext, one method involves using PBS containing 20% DMSO and 8% humanserum albumin, or other suitable cell freezing media, the cells then arefrozen to −80° C. at a rate of 1° per minute and stored in the vaporphase of a liquid nitrogen storage tank. Other methods of controlledfreezing may be used as well as uncontrolled freezing immediately at−20° C. or in liquid nitrogen.

T cells for use in the present invention may also be antigen-specific Tcells. For example, tumor-specific T cells can be used. In certainembodiments, antigen-specific T cells can be isolated from a patient ofinterest, such as a patient afflicted with a cancer or an infectiousdisease. In one embodiment, neoepitopes are determined for a subject andT cells specific to these antigens are isolated. Antigen-specific cellsfor use in expansion may also be generated in vitro using any number ofmethods known in the art, for example, as described in U.S. PatentPublication No. US 20040224402 entitled, Generation and Isolation ofAntigen-Specific T Cells, or in U.S. Pat. No. 6,040,177.Antigen-specific cells for use in the present invention may also begenerated using any number of methods known in the art, for example, asdescribed in Current Protocols in Immunology, or Current Protocols inCell Biology, both published by John Wiley & Sons, Inc., Boston, Mass.

In a related embodiment, it may be desirable to sort or otherwisepositively select (e.g. via magnetic selection) the antigen specificcells prior to or following one or two rounds of expansion. Sorting orpositively selecting antigen-specific cells can be carried out usingpeptide-MIIC tetramers (Altman, et al., Science. 1996 Oct. 4;274(5284):94-6). In another embodiment, the adaptable tetramertechnology approach is used (Andersen et al., 2012 Nat Protoc.7:891-902). Tetramers are limited by the need to utilize predictedbinding peptides based on prior hypotheses, and the restriction tospecific HLAs. Peptide-MHIC tetramers can be generated using techniquesknown in the art and can be made with any MIIC molecule of interest andany antigen of interest as described herein. Specific epitopes to beused in this context can be identified using numerous assays known inthe art. For example, the ability of a polypeptide to bind to MIIC classI may be evaluated indirectly by monitoring the ability to promoteincorporation of ¹²⁵I labeled p2-microglobulin (02m) into MHC classI/β2m/peptide heterotrimeric complexes (see Parker et al., J. Immunol.152:163, 1994).

In one embodiment cells are directly labeled with an epitope-specificreagent for isolation by flow cytometry followed by characterization ofphenotype and TCRs. In one embodiment, T cells are isolated bycontacting with T cell specific antibodies. Sorting of antigen-specificT cells, or generally any cells of the present invention, can be carriedout using any of a variety of commercially available cell sorters,including, but not limited to, MoFlo sorter (DakoCytomation, FortCollins, Colo.), FACSAria™, FACSArray™, FACSVantage™, BD™ LSR II, andFACSCalibur™ (BD Biosciences, San Jose, Calif.).

In a preferred embodiment, the method comprises selecting cells thatalso express CD3. The method may comprise specifically selecting thecells in any suitable manner. Preferably, the selecting is carried outusing flow cytometry. The flow cytometry may be carried out using anysuitable method known in the art. The flow cytometry may employ anysuitable antibodies and stains. Preferably, the antibody is chosen suchthat it specifically recognizes and binds to the particular biomarkerbeing selected. For example, the specific selection of CD3, CD8, TIM-3,LAG-3, 4-1BB, or PD-1 may be carried out using anti-CD3, anti-CD8,anti-TIM-3, anti-LAG-3, anti-4-1BB, or anti-PD-1 antibodies,respectively. The antibody or antibodies may be conjugated to a bead(e.g., a magnetic bead) or to a fluorochrome. Preferably, the flowcytometry is fluorescence-activated cell sorting (FACS). TCRs expressedon T cells can be selected based on reactivity to autologous tumors.Additionally, T cells that are reactive to tumors can be selected forbased on markers using the methods described in patent publication Nos.WO2014133567 and WO2014133568, herein incorporated by reference in theirentirety. Additionally, activated T cells can be selected for based onsurface expression of CD107a.

In one embodiment of the invention, the method further comprisesexpanding the numbers of T cells in the enriched cell population. Suchmethods are described in U.S. Pat. No. 8,637,307 and is hereinincorporated by reference in its entirety. The numbers of T cells may beincreased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), morepreferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-,or 90-fold), more preferably at least about 100-fold, more preferably atleast about 1,000 fold, or most preferably at least about 100,000-fold.The numbers of T cells may be expanded using any suitable method knownin the art. Exemplary methods of expanding the numbers of cells aredescribed in patent publication No. WO 2003057171, U.S. Pat. No.8,034,334, and U.S. Patent Application Publication No. 2012/0244133,each of which is incorporated herein by reference.

In one embodiment, ex vivo T cell expansion can be performed byisolation of T cells and subsequent stimulation or activation followedby further expansion. In one embodiment of the invention, the T cellsmay be stimulated or activated by a single agent. In another embodiment,T cells are stimulated or activated with two agents, one that induces aprimary signal and a second that is a co-stimulatory signal. Ligandsuseful for stimulating a single signal or stimulating a primary signaland an accessory molecule that stimulates a second signal may be used insoluble form. Ligands may be attached to the surface of a cell, to anEngineered Multivalent Signaling Platform (EMSP), or immobilized on asurface. In a preferred embodiment both primary and secondary agents areco-immobilized on a surface, for example a bead or a cell. In oneembodiment, the molecule providing the primary activation signal may bea CD3 ligand, and the co-stimulatory molecule may be a CD28 ligand or4-1BB ligand.

In certain embodiments, T cells comprising a CAR or an exogenous TCR,may be manufactured as described in WO2015120096, by a methodcomprising: enriching a population of lymphocytes obtained from a donorsubject; stimulating the population of lymphocytes with one or moreT-cell stimulating agents to produce a population of activated T cells,wherein the stimulation is performed in a closed system using serum-freeculture medium; transducing the population of activated T cells with aviral vector comprising a nucleic acid molecule which encodes the CAR orTCR, using a single cycle transduction to produce a population oftransduced T cells, wherein the transduction is performed in a closedsystem using serum-free culture medium; and expanding the population oftransduced T cells for a predetermined time to produce a population ofengineered T cells, wherein the expansion is performed in a closedsystem using serum-free culture medium. In certain embodiments, T cellscomprising a CAR or an exogenous TCR, may be manufactured as describedin WO2015120096, by a method comprising: obtaining a population oflymphocytes; stimulating the population of lymphocytes with one or morestimulating agents to produce a population of activated T cells, whereinthe stimulation is performed in a closed system using serum-free culturemedium; transducing the population of activated T cells with a viralvector comprising a nucleic acid molecule which encodes the CAR or TCR,using at least one cycle transduction to produce a population oftransduced T cells, wherein the transduction is performed in a closedsystem using serum-free culture medium; and expanding the population oftransduced T cells to produce a population of engineered T cells,wherein the expansion is performed in a closed system using serum-freeculture medium. The predetermined time for expanding the population oftransduced T cells may be 3 days. The time from enriching the populationof lymphocytes to producing the engineered T cells may be 6 days. Theclosed system may be a closed bag system. Further provided is populationof T cells comprising a CAR or an exogenous TCR obtainable or obtainedby said method, and a pharmaceutical composition comprising such cells.

In certain embodiments, T cell maturation or differentiation in vitromay be delayed or inhibited by the method as described in WO2017070395,comprising contacting one or more T cells from a subject in need of a Tcell therapy with an AKT inhibitor (such as, e.g., one or a combinationof two or more AKT inhibitors disclosed in claim 8 of WO2017070395) andat least one of exogenous Interleukin-7 (IL-7) and exogenousInterleukin-15 (IL-15), wherein the resulting T cells exhibit delayedmaturation or differentiation, and/or wherein the resulting T cellsexhibit improved T cell function (such as, e.g., increased T cellproliferation; increased cytokine production; and/or increased cytolyticactivity) relative to a T cell function of a T cell cultured in theabsence of an AKT inhibitor.

In certain embodiments, a patient in need of a T cell therapy may beconditioned by a method as described in WO2016191756 comprisingadministering to the patient a dose of cyclophosphamide between 200mg/m2/day and 2000 mg/m2/day and a dose of fludarabine between 20mg/m2/day and 900 mg/m²/day.

In some embodiments, the cancer cells in the subject to be treatedexpress CLECL2D. In some embodiments, the immune cells in the tumormicroenvironment express KLRB1. As such, they may be tumor infiltratinglymphocytes. In certain embodiments, the cancer expresses CLEC2D, orcells in the cancer express CLEC2D. In certain embodiments, tumorinfiltrating lymphocytes (TILs) in the cancer express KLRB1.

The terms “cancer” or “tumor” or “hyperproliferative disorder” refer tothe presence of cells possessing characteristics typical ofcancer-causing cells, such as uncontrolled proliferation, immortality,metastatic potential, rapid growth and proliferation rate, and certaincharacteristic morphological features. Cancer cells are often in theform of a tumor, but such cells may exist alone within an animal, or maybe a non-tumorigenic cancer cell, such as a leukemia cell. Cancersinclude, but are not limited to, B cell cancer, e.g., multiple myeloma,Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, forexample, alpha chain disease, gamma chain disease, and mu chain disease,benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas,breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostatecancer (e.g., metastatic, hormone refractory prostate cancer),pancreatic cancer, stomach cancer, ovarian cancer, urinary bladdercancer, brain or central nervous system cancer, peripheral nervoussystem cancer, esophageal cancer, cervical cancer, uterine orendometrial cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, cancer of hematologicaltissues, and the like. Other non-limiting examples of types of cancersapplicable to the methods encompassed by the present invention includehuman sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer,breast cancer, ovarian cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, liver cancer,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, bone cancer, brain tumor, testicular cancer, lung carcinoma,small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g.,acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronicleukemia (chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, and heavy chain disease. In some embodiments, thecancer whose phenotype is determined by the method of the presentinvention is an epithelial cancer such as, but not limited to, bladdercancer, breast cancer, cervical cancer, colon cancer, gynecologiccancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, headand neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, orskin cancer. In other embodiments, the cancer is breast cancer,prostrate cancer, lung cancer, or colon cancer. In still otherembodiments, the epithelial cancer is non-small-cell lung cancer,nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma(e.g., serous ovarian carcinoma), or breast carcinoma. The epithelialcancers may be characterized in various other ways including, but notlimited to, serous, endometrioid, mucinous, clear cell, brenner, orundifferentiated. In some embodiments, the present invention is used inthe treatment, diagnosis, and/or prognosis of lymphoma or its subtypes,including, but not limited to, mantle cell lymphoma.

In certain embodiments, the cancer is glioblastoma multiforme (GBM). GBMis an aggressive, primary tumor of the central nervous system (Furnariet al. (2007) Genes Dev. 21:2683-7270). Because of their intrinsic,infiltrative nature, GBMs follow a malignant clinical course. Classifiedas World Health Organization grade IV astrocytic tumors, GBMs have apronounced mitotic activity, substantial tendency toward neoangiogenesis(microvascular proliferation), necrosis, and proliferative rates threeto five times higher than grade III tumors, the anaplastic astrocytomas.The clinical behavior of GBMs is often mimicked by unusual pathologicalpresentations, which gave rise to the old moniker of “glioblastomamultiforme.” Even with the survival advantage provided by the recentlydeveloped protocol of concurrent chemoradiation followed by adjuvantalkylating chemotherapy with temozolomide (the Stupp regimen), theprognosis of patients with GBM remains poor with median overall survivalin the range of 9-15 months and two-year survival rates of 26% in themost favorable subgroup (Stupp et al. (2005) N. Engl. J. Med.352:987-996). The outlook for patients with malignant gliomas is poor.Median survival for patients with moderately severe (grade III)malignant gliomas is three to five years. For patients with the mostsevere, aggressive form of malignant glioma (grade IV glioma orglioblastoma multiforme), median survival is less than a year.

In some embodiments, the cancer to be treated may be glioblastomamultiforme (GBM), renal cancer, lung adenocarcinoma, or colonadenocarcinoma.

Isolated T Cells, Populations Thereof, and Compositions

The disclosure also provides methods of treating cancer in a subject inneed thereof by administering a pharmaceutical composition comprising apopulation of T cells as described in more detail below.

Provided herein are isolated T cells or populations of such T cells thatare modified to exhibit decreased expression or activity of, or, aremodified to have an agent that is capable of decreasing expression oractivity of KLRB1. Such T cells or populations thereof may be CD8+ Tcells or CD4+ T cells. In embodiments, the T cell or population thereofmay be obtained from peripheral blood mononuclear cells (PBMCs). The Tcell or population thereof may be an autologous T cell from a subject inneed of treatment. In some embodiments, the T cell or population thereofmay be a tumor TIL obtained from a subject in need of treatment. The Tcell or population thereof may have a chimeric antigen receptor (CAR) oran exogenous T-cell receptor (TCR) as described below. In someembodiments, the exogenous TCR may be clonally expanded in a tumor. Insome embodiments, the CAR or TCR may be specific for a tumor antigen. Inspecific embodiments, the tumor antigen may be EGFRvIII.

Alternatively, the tumor antigen may be selected from the groupconsisting of: B cell maturation antigen (BCMA); PSA (prostate-specificantigen); prostate-specific membrane antigen (PSMA); PSCA (Prostate stemcell antigen); Tyrosine-protein kinase transmembrane receptor ROR1;fibroblast activation protein (FAP); Tumor-associated glycoprotein 72(TAG72); Carcinoembryonic antigen (CEA); Epithelial cell adhesionmolecule (EPCAM); Mesothelin; Human Epidermal growth factor Receptor 2(ERBB2 (Her2/neu)); Prostase; Prostatic acid phosphatase (PAP);elongation factor 2 mutant (ELF2M); Insulin-like growth factor 1receptor (IGF-1R); gplOO; BCR-ABL (breakpoint cluster region-Abelson);tyrosinase; New York esophageal squamous cell carcinoma 1 (NY-ESO—1);x-light chain, LAGE (L antigen); MAGE (melanoma antigen);Melanoma-associated antigen 1 (MAGE-A1); MAGE A3; MAGE A6; legumain;Human papillomavirus (HPV) E6; HPV E7; prostein; survivin; PCTA1(Galectin 8); Melan-A/MART-1; Ras mutant; TRP-1 (tyrosinase relatedprotein 1, or gp75); Tyrosinase-related Protein 2 (TRP2); TRP-2/INT2(TRP-2/intron 2); RAGE (renal antigen); receptor for advanced glycationend products 1 (RAGE1); Renal ubiquitous 1, 2 (RU1, RU2); intestinalcarboxyl esterase (iCE); Heat shock protein 70-2 (HSP70-2) mutant;thyroid stimulating hormone receptor (TSHR); CD123; CD171; CD19; CD20;CD22; CD26; CD30; CD33; CD44v7/8 (cluster of differentiation 44, exons7/8); CD53; CD92; CD100; CD148; CD150; CD200; CD261; CD262; CD362; CS-1(CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-likemolecule-1 (CLL-1); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (Tn Ag);Fms-Like Tyrosine Kinase 3 (FLT3); CD38; CD138; CD44v6; B7H3 (CD276);KIT (CD 117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2);Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen(PSCA); Protease Serine 21 (PRSS21); vascular endothelial growth factorreceptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growthfactor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4(SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16 (MUC16);epidermal growth factor receptor (EGFR); epidermal growth factorreceptor variant III (EGFRvIII); neural cell adhesion molecule (NCAM);carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit,Beta Type, 9 (LMP2); ephrin type-A receptor 2 (EphA2); Ephrin B2;Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; high molecularweight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside(OAcGD2); Folate receptor alpha; Folate receptor beta; tumor endothelialmarker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R);claudin 6 (CLDN6); G protein-coupled receptor class C group 5, member D(GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a;anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1(PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH);mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2);Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3(ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2(OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumorprotein (WT1); ETS translocation-variant gene 6, located on chromosome12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A(XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); CT(cancer/testis (antigen)); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53;p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin Bi; Cyclin Di; v-myc avian myelocytomatosisviral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog FamilyMember C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor(Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma AntigenRecognized By T Cells-1 or 3 (SART1, SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint-1, -2, -3 or -4 (SSX1, SSX2, SSX3, SSX4);CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1(LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300molecule-like family member f (CD300LF); C-type lectin domain family 12member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyteantigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); mousedouble minute 2 homolog (MDM2); livin; alphafetoprotein (AFP);transmembrane activator and CAML Interactor (TACI); B-cell activatingfactor receptor (BAFF-R); V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog (KRAS); immunoglobulin lambda-like polypeptide 1 (IGLL1); 707-AP(707 alanine proline); ART-4 (adenocarcinoma antigen recognized by T4cells); BAGE (B antigen; b-catenin/m, b-catenin/mutated); CAMEL(CTL-recognized antigen on melanoma); CAPi (carcinoembryonic antigenpeptide 1); CASP-8 (caspase-8); CDCl27m (cell-division cycle 27mutated); CDK4/m (cycline-dependent kinase 4 mutated); Cyp-B(cyclophilin B); DAM (differentiation antigen melanoma); EGP-2(epithelial glycoprotein 2); EGP-40 (epithelial glycoprotein 40); Erbb2,3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4); FBP(folate binding protein); , fAchR (Fetal acetylcholine receptor); G250(glycoprotein 250); GAGE (G antigen); GnT-V(N-acetylglucosaminyltransferase V); HAGE (helicose antigen); ULA-A(human leukocyte antigen-A); HST2 (human signet ring tumor 2); KIAA0205;KDR (kinase insert domain receptor); LDLR/FUT (low density lipidreceptor/GDP L-fucose: b-D-galactosidase 2-a-L fucosyltransferase);L1CAM (L1 cell adhesion molecule); MC1R (melanocortin 1 receptor);Myosin/m (myosin mutated); MUM-1, -2, -3 (melanoma ubiquitous mutated 1,2, 3); NA88-A (NA cDNA clone of patient M88); KG2D (Natural killer group2, member D) ligands; oncofetal antigen (h5T4); p190 minor bcr-abl(protein of 190KD bcr-abl); Pml/RARa (promyelocytic leukaemia/retinoicacid receptor a); PRAME (preferentially expressed antigen of melanoma);SAGE (sarcoma antigen); TEL/AML1 (translocation Ets-familyleukemia/acute myeloid leukemia 1); TPI/m (triosephosphate isomerasemutated); and any combination thereof.

The T cell or population thereof may be further modified to havedecreased expression or activity of, or may be modified to have an agentcapable of decreasing expression or activity of a gene or polypeptideselected from the group consisting of TOB1, RGS1, TARP, NKG7, CCL4 andany combination thereof. The T cell or T cells within such a populationmay be activated. The T cell or population thereof may be modified usinga CRISPR system having guide sequences specific to the target, asdescribed in more detail below. The CRISPR system may include, but notbe necessarily limited to, Cas9, Cpf1 or Cas13 as described in detailabove.

The disclosure also provides methods of generating a population of Tcells for adoptive cell transfer by a) obtaining a population of Tcells; b) delivering to the population of T cells a CRISPR systemcomprising one or more guide sequences targeting KLRB1; and c)activating the population of cells. The T cells may be obtained fromTILs obtained from a subject in need of treatment. In certainembodiments, activating involves culturing the population of cells withαCD3 and αCD28 beads and IL-2. In further embodiments, the population ofcells may be transduced with a vector encoding a chimeric antigenreceptor (CAR) or an exogenous T-cell receptor (TCR). The vector mayfurther encode a detectable marker and the T cells expressing a CAR orTCR may be purified by sorting cells positive for the detectable marker.The T cells may be obtained from PBMCs. In embodiments, the PBMCs may beobtained from a subject in need of treatment.

Populations of T cells as described herein may be comprised in apharmaceutical composition, as described in detail above. In specificembodiments, the composition may be administered to a subject in needthereof by any suitable means described herein.

In specific embodiments, the pollution of cells may be administered byinfusion into the cerebral spinal fluid (CSF) as described herein. Insome embodiments, this administration may be carried out by injectionthrough the lateral ventricle. In some embodiments, the population ofcells is administered in a combination treatment regimen comprisingcheckpoint blockade therapy as described herein. In some embodiments,the checkpoint blockade therapy comprises anti-PD-1, anti-CTLA4,anti-PDL1, anti-TIM-3 and/or anti-LAG3. In some embodiments, the cancerexpresses CLEC2D as described herein. In some embodiments, the tumorinfiltrating lymphocytes (TILs) in the cancer express KLRB1. In someembodiments, the cancer is glioblastoma multiforme (GBM) as describedherein.

Methods of Treating Cancer

The agents and compositions described herein may be used in thetreatment of several disease types. In certain example embodiments, theagents and compositions described herein may be used to treat cancer.

In some embodiments, the disclosure provides methods of treating cancerin a subject in need thereof by administering an agent capable ofbinding the KLRB1 receptor on tumor-infiltrating T lymphocytes. In otherembodiments, the methods involve treating cancer in a subject in needthereof by administering an agent capable of binding a KLRB1 ligand. Inone embodiment, that ligand may include, but is not necessarily limitedto, CLEC2D. In some embodiments, methods involve administering an agentcapable of blocking the interaction of KLRB1 with its ligand.

As used in this context, to “treat” means to cure, ameliorate,stabilize, prevent, or reduce the severity of at least one symptom or adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder. It is understood that treatment, while intendedto cure, ameliorate, stabilize, or prevent a disease, pathologicalcondition, or disorder, need not actually result in the cure,amelioration, stabilization or prevention. The effects of treatment canbe measured or assessed as described herein and as known in the art asis suitable for the disease, pathological condition, or disorderinvolved. Such measurements and assessments can be made in qualitativeand/or quantitative terms. Thus, for example, characteristics orfeatures of a disease, pathological condition, or disorder and/orsymptoms of a disease, pathological condition, or disorder can bereduced to any effect or to any amount.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver (e.g. physician, nurse, nurse practitioner, or individualin the case of humans; veterinarian in the case of animals, includingnon-human animals) that a subject requires or will benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a caregiver's experience, but that include the knowledgethat the subject is ill, or will be ill, as the result of a conditionthat is treatable by the compositions and therapeutic agents describedherein.

The administration of compositions, agents, cells, or populations ofcells, as disclosed herein may be carried out in any convenient mannerincluding by aerosol inhalation, injection, ingestion, transfusion,implantation or transplantation. The cells or population of cells may beadministered to a patient subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, intrathecally, byintravenous or intralymphatic injection, or intraperitoneally. In someembodiments, the disclosed CARs may be delivered or administered into acavity formed by the resection of tumor tissue (i.e. intracavitydelivery) or directly into a tumor prior to resection (i.e. intratumoraldelivery). In one embodiment, the population of cells are administeredby infusion into the cerebral spinal fluid (CSF), preferably through thelateral ventricle.

Tumor-infiltrating lymphocytes are white blood cells that have left thebloodstream and migrated toward a tumor. They include T cells and Bcells and are part of the larger category of ‘tumor-infiltrating immunecells’, which consist of both mononuclear and polymorphonuclear immunecells, such as T cells, B cells, natural killer cells, macrophages,neutrophils, dendritic cells, mast cells, eosinophils, basophils, etc.,in variable proportions. Their abundance varies in different types oftumors and stages and in some cases relate to disease prognosis.

Methods of Treating Infectious Diseases

The disclosure also provides methods of treating infectious diseases byadministering an agent capable of binding the KLRB1 receptor on T cells,or by blocking the interaction of KLRB1 with its ligand as describedherein.

Infectious diseases may be caused by a variety of microbes which includebacteria, fungi, protozoa, parasites, and viruses.

Bacterial diseases may include diseases caused by any one or more of (orany combination of) Acinetobacter baumanii, Actinobacillus sp.,Actinomycetes, Actinomyces sp. (such as Actinomyces israelii andActinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila,Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonascaviae), Anaplasma phagocytophilum, Anaplasma marginale Alcaligenesxylosoxidans, Acinetobacter baumanii, Actinobacillusactinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis,Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillusstearothermophilus), Bacteroides sp. (such as Bacteroides fragilis),Bartonella sp. (such as Bartonella bacilliformis and Bartonellahenselae, Bifidobacterium sp., Bordetella sp. (such as Bordetellapertussis, Bordetella parapertussis, and Bordetella bronchiseptica),Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi),Brucella sp. (such as Brucella abortus, Brucella canis, Brucellamelintensis and Brucella suis), Burkholderia sp. (such as Burkholderiapseudomallei and Burkholderia cepacia), Campylobacter sp. (such asCampylobacter jejuni, Campylobacter coli, Campylobacter lari andCampylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis,Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci,Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as,Corynebacterium diphtheriae, Corynebacterium jeikeum andCorynebacterium), Clostridium sp. (such as Clostridium perfringens,Clostridium difficile, Clostridium botulinum and Clostridium tetani),Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes,Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli,including opportunistic Escherichia coli, such as enterotoxigenic E.coli, enteroinvasive E. coli, enteropathogenic E. coli,enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenicE. coli) Enterococcus sp. (such as Enterococcus faecalis andEnterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia andEhrlichia canis), Epidermophyton floccosum, Erysipelothrixrhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacteriumnucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp.(such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilusaegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus andHaemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacterpylori, Helicobacter cinaedi and Helicobacter fennelliae), Kingellakingii, Klebsiella sp. (such as Klebsiella pneumoniae, Klebsiellagranulomatis and Klebsiella oxytoca), Lactobacillus sp., Listeriamonocytogenes, Leptospira interrogans, Legionella pneumophila,Leptospira interrogans, Peptostreptococcus sp., Mannheimia hemolytica,Microsporum canis, Moraxella catarrhalis, Morganella sp., Mobiluncussp., Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae,Mycobacterium tuberculosis, Mycobacterium paratuberculosis,Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis,and Mycobacterium marinum), Mycoplasm sp. (such as Mycoplasmapneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp.(such as Nocardia asteroides, Nocardia cyriacigeorgica and Nocardiabrasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae andNeisseria meningitidis), Pasteurella multocida, Pityrosporum orbiculare(Malassezia furfur), Plesiomonas shigelloides. Prevotella sp.,Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such asProteus vulgaris and Proteus mirabilis), Providencia sp. (such asProvidencia alcalifaciens, Providencia rettgeri and Providenciastuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcusequi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akariand Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsiatsutsugamushi) and Rickettsia typhi), Rhodococcus sp., Serratiamarcescens, Stenotrophomonas maltophilia, Salmonella sp. (such asSalmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Salmonella cholerasuis and Salmonella typhimurium),Serratia sp. (such as Serratia marcesans and Serratia liquifaciens),Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigellaboydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus hemolyticus,Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcuspneumoniae (for example chloramphenicol-resistant serotype 4Streptococcus pneumoniae, spectinomycin-resistant serotype 6BStreptococcus pneumoniae, streptomycin-resistant serotype 9VStreptococcus pneumoniae, erythromycin-resistant serotype 14Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcuspneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae,tetracycline-resistant serotype 19F Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, andtrimethoprim-resistant serotype 23F Streptococcus pneumoniae,chloramphenicol-resistant serotype 4 Streptococcus pneumoniae,spectinomycin-resistant serotype 6B Streptococcus pneumoniae,streptomycin-resistant serotype 9V Streptococcus pneumoniae,optochin-resistant serotype 14 Streptococcus pneumoniae,rifampicin-resistant serotype 18C Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, ortrimethoprim-resistant serotype 23F Streptococcus pneumoniae),Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes,Group A streptococci, Streptococcus pyogenes, Group B streptococci,Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus,Streptococcus equismilis, Group D streptococci, Streptococcus bovis,Group F streptococci, and Streptococcus anginosus Group G streptococci),Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such asTreponema carateum, Treponema petenue, Treponema pallidum and Treponemaendemicum, Trichophyton rubrum, T. mentagrophytes, Tropheryma whippelii,Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibriocholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrioparahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibriomimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibriodamsela and Vibrio furnisii), Yersinia sp. (such as Yersiniaenterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis) andXanthomonas maltophilia among others.

Fungal diseases may include diseases caused by any one or more of (orany combination of) Aspergillus, Blastomyces, Candidiasis,Coccidiodomycosis, Cryptococcus neoformans, Cryptococcus gatti, sp.Histoplasma sp. (such as Histoplasma capsulatum), Pneumocystis sp. (suchas Pneumocystis jirovecii), Stachybotrys (such as Stachybotryschartarum), Mucroymcosis, Sporothrix, fungal eye infections ringworm,Exserohilum, Cladosporium.

In certain example embodiments, the fungus is a yeast. Examples of yeastthat can be detected in accordance with disclosed methods includewithout limitation one or more of (or any combination of), Aspergillusspecies (such as Aspergillus fumigatus, Aspergillus flavus andAspergillus clavatus), Cryptococcus sp. (such as Cryptococcusneoformans, Cryptococcus gattii, Cryptococcus laurentii and Cryptococcusalbidus), a Geotrichum species, a Saccharomyces species, a Hansenulaspecies, a Candida species (such as Candida albicans), a Kluyveromycesspecies, a Debaryomyces species, a Pichia species, or combinationthereof. In certain example embodiments, the fungi is a mold. Examplemolds include, but are not limited to, a Penicillium species, aCladosporium species, a Byssochlamys species, or a combination thereof.

Protozoan diseases may include diseases caused by any one or more of (orany combination of) Euglenozoa, Heterolobosea, Diplomonadida, Amoebozoa,Blastocystic, and Apicomplexa. Example Euglenoza include, but are notlimited to, Trypanosoma cruzi (Chagas disease), T. brucei gambiense, T.brucei rhodesiense, Leishmania braziliensis, L. infantum, L. mexicana,L. major, L. tropica, and L. donovani. Example Heterolobosea include,but are not limited to, Naegleria fowleri. Example Diplomonadidsinclude, but are not limited to, Giardia intestinalis (G. lamblia, G.duodenalis). Example Amoebozoa include, but are not limited to,Acanthamoeba castellanii, Balamuthia madrillaris, Entamoeba histolytica.Example Blastocysts include, but are not limited to, Blastocystichominis. Example Apicomplexa include, but are not limited to, Babesiamicroti, Cryptosporidium parvum, Cyclospora cayetanensis, Plasmodiumfalciparum, P. vivax, P. ovale, P. malariae, and Toxoplasma gondii.

Parasitic diseases may include, but are not necessarily limited to,diseases caused by any one or more of (or any combination of) Onchocercaspecies and a Plasmodium species. In specific embodiments, the diseasecause by a parasitic infection is malaria.

Viral diseases may include diseases caused by any one or more of (or anycombination of) Ebolavirus, measles virus, SARS, Chikungunya virus,hepatitis viruses, Marburg virus, yellow fever virus, MERS, Denguevirus, Lassa fever virus, influenza virus, rhabdovirus or HIV. Ahepatitis virus may include hepatitis A, hepatitis B, or hepatitis C. Aninfluenza virus may include, for example, influenza A or influenza B. AnHIV may include HIV 1 or HIV 2. In certain example embodiments, theviral sequence may be a human respiratory syncytial virus, Sudan ebolavirus, Bundibugyo virus, Tai Forest ebola virus, Reston ebola virus,Achimota, Aedes flavivirus, Aguacate virus, Akabane virus, Alethinophidreptarenavirus, Allpahuayo mammarenavirus, Amapari mmarenavirus, Andesvirus, Apoi virus, Aravan virus, Aroa virus, Arumwot virus, Atlanticsalmon paramyxovirus, Australian bat lyssavirus, Avian bornavirus, Avianmetapneumovirus, Avian paramyxoviruses, penguin or FalklandIslandsvirus, BK polyomavirus, Bagaza virus, Banna virus, Batherpesvirus, Bat sapovirus, Bear Canon mammarenavirus, Beilong virus,Betacoronavirus, Betapapillomavirus 1-6, Bhanja virus, Bokeloh batlyssavirus, Borna disease virus, Bourbon virus, Bovine hepacivirus,Bovine parainfluenza virus 3, Bovine respiratory syncytial virus,Brazoran virus, Bunyamwera virus, Caliciviridae virus. Californiaencephalitis virus, Candiru virus, Canine distemper virus, Caninepneumovirus, Cedar virus, Cell fusing agent virus, Cetaceanmorbillivirus, Chandipura virus, Chaoyang virus, Chapare mammarenavirus,Chikungunya virus, Colobus monkey papillomavirus, Colorado tick fevervirus, Cowpox virus, Crimean-Congo hemorrhagic fever virus, Culexflavivirus, Cupixi mammarenavirus, Dengue virus, Dobrava-Belgrade virus,Donggang virus, Dugbe virus, Duvenhage virus, Eastern equineencephalitis virus, Entebbe bat virus, Enterovirus A-D, European batlyssavirus 1-2, Eyach virus, Feline morbillivirus, Fer-de-Lanceparamyxovirus, Fitzroy River virus, Flaviviridae virus, Flexalmammarenavirus, GB virus C, Gairo virus, Gemycircularvirus, Gooseparamyxovirus SF02, Great Island virus, Guanarito mammarenavirus,Hantaan virus, Hantavirus Z10, Heartland virus, Hendra virus, HepatitisA/B/C/E, Hepatitis delta virus, Human bocavirus, Human coronavirus,Human endogenous retrovirus K, Human enteric coronavirus, Humangenital-associated circular DNA virus-1, Human herpesvirus 1-8, Humanimmunodeficiency virus 1/2, Human mastadenovirus A-G, Humanpapillomavirus, Human parainfluenza virus 1-4, Human paraechovirus,Human picornavirus, Human smacovirus, Ikoma lyssavirus, Ilheus virus,Influenza A-C, Ippy mammarenavirus, Irkut virus, J-virus, JCpolyomavirus, Japanese encephalitis virus, Junin mammarenavirus, KIpolyomavirus, Kadipiro virus, Kamiti River virus, Kedougou virus,Khujand virus, Kokobera virus, Kyasanur forest disease virus, Lagos batvirus, Langat virus, Lassa mammarenavirus, Latino mammarenavirus,Leopards Hill virus, Liao ning virus, Ljungan virus, Lloviu virus,Louping ill virus, Lujo mammarenavirus, Luna mammarenavirus, Lunk virus,Lymphocytic choriomeningitis mammarenavirus, Lyssavirus Ozernoe,MSSI2\225 virus, Machupo mammarenavirus, Mamastrovirus 1, Manzanillavirus, Mapuera virus, Marburg virus, Mayaro virus, Measles virus,Menangle virus, Mercadeo virus, Merkel cell polyomavirus, Middle Eastrespiratory syndrome coronavirus, Mobala mammarenavirus, Modoc virus,Moijang virus, Mokolo virus, Monkeypox virus, Montana myotisleukoenchalitis virus, Mopeia lassa virus reassortant 29, Mopeiamammarenavirus, Morogoro virus, Mossman virus, Mumps virus, Murinepneumonia virus, Murray Valley encephalitis virus, Nariva virus,Newcastle disease virus, Nipah virus, Norwalk virus, Norway rathepacivirus, Ntaya virus, O'nyong-nyong virus, Oliveros mammarenavirus,Omsk hemorrhagic fever virus, Oropouche virus, Parainfluenza virus 5,Parana mammarenavirus, Parramatta River virus,Peste-des-petits-ruminants virus, Pichande mammarenavirus,Picornaviridae virus, Pirital mammarenavirus, Piscihepevirus A, Porcineparainfluenza virus 1, porcine rubulavirus, Powassan virus, PrimateT-lymphotropic virus 1-2, Primate erythroparvovirus 1, Punta Toro virus,Puumala virus, Quang Binh virus, Rabies virus, Razdan virus, Reptilebornavirus 1, Rhinovirus A-B, Rift Valley fever virus, Rinderpest virus,Rio Bravo virus, Rodent Torque Teno virus, Rodent hepacivirus, RossRiver virus, Rotavirus A-I, Royal Farm virus, Rubella virus, Sabiamammarenavirus, Salem virus, Sandfly fever Naples virus, Sandfly feverSicilian virus, Sapporo virus, Sathuperi virus, Seal anellovirus,Semliki Forest virus, Sendai virus, Seoul virus, Sepik virus, Severeacute respiratory syndrome-related coronavirus, Severe fever withthrombocytopenia syndrome virus, Shamonda virus, Shimoni bat virus,Shuni virus, Simbu virus, Simian torque teno virus, Simian virus 40-41,Sin Nombre virus, Sindbis virus, Small anellovirus, Sosuga virus,Spanish goat encephalitis virus, Spondweni virus, St. Louis encephalitisvirus, Sunshine virus, TTV-like mini virus, Tacaribe mammarenavirus,Taila virus, Tamana bat virus, Tamiami mammarenavirus, Tembusu virus,Thogoto virus, Thottapalayam virus, Tick-borne encephalitis virus,Tioman virus, Togaviridae virus, Torque teno canis virus, Torque tenodouroucouli virus, Torque teno felis virus, Torque teno midi virus,Torque teno sus virus, Torque teno tamarin virus, Torque teno virus,Torque teno zalophus virus, Tuhoko virus, Tula virus, Tupaiaparamyxovirus, Usutu virus, Uukuniemi virus, Vaccinia virus, Variolavirus, Venezuelan equine encephalitis virus, Vesicular stomatitisIndiana virus, WU Polyomavirus, Wesselsbron virus, West Caucasian batvirus, West Nile virus, Western equine encephalitis virus, WhitewaterArroyo mammarenavirus, Yellow fever virus, Yokose virus, Yug Bogdanovacvirus, Zaire ebolavirus, Zika virus, or Zygosaccharomyces bailii virus Zviral sequence. Examples of RNA viruses that may be detected include oneor more of (or any combination of) Coronaviridae virus, a Picornaviridaevirus, a Caliciviridae virus, a Flaviviridae virus, a Togaviridae virus,a Bornaviridae, a Filoviridae, a Paramyxoviridae, a Pneumoviridae, aRhabdoviridae, an Arenaviridae, a Bunyaviridae, an Orthomyxoviridae, ora Deltavirus. In certain example embodiments, the virus is Coronavirus,SARS, Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, Yellow fevervirus, West Nile virus, Hepatitis C virus, Dengue fever virus, Zikavirus, Rubella virus, Ross River virus, Sindbis virus, Chikungunyavirus, Borna disease virus, Ebola virus, Marburg virus, Measles virus,Mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, Humanrespiratory syncytial virus, Rabies virus, Lassa virus, Hantavirus,Crimean-Congo hemorrhagic fever virus, Influenza, or Hepatitis D virus.

In certain example embodiments, the virus may be a retrovirus. Exampleretroviruses that may be detected using the embodiments disclosed hereininclude one or more of or any combination of viruses of the GenusAlpharetrovirus, Betaretrovirus, Gammaretrovirus, Deltaretrovirus,Epsilonretrovirus, Lentivirus, Spumavirus, or the Family Metaviridae,Pseudoviridae, and Retroviridae (including HIV), Hepadnaviridae(including Hepatitis B virus), and Caulimoviridae (including Cauliflowermosaic virus).

In some embodiments, the infectious diseases comprises a chronic viralinfection, such as HIV. In some embodiments, the infectious diseasecomprises a chronic bacterial infection such as tuberculosis (TB). Insome embodiments, the infectious disease comprises a chronic parasiticinfection such as malaria.

In methods of treating the infectious disease, the agent may comprise anantibody or a fragment thereof. In specific embodiments, the antibodybinds KLRB1 as described herein. In specific embodiments, the antibodybinds CLEC2D as described herein.

In specific embodiments, the method comprises treating one or morebacterial infections of the intestine in a subject in need thereof byadministering an agent capable of blocking the interaction of KLRB1 withis ligand. As such, this blocking may enhance the function of mucosalassociated invariant T (MAIT) cells.

MAIT cells make up a subset of T cells in the immune system that displayinnate, effector-like qualities. In humans, MAIT cells are found in theblood, liver, lungs, and mucosa, defending against microbial activityand infection. They constitute a subset of ap T lymphocytescharacterized by a semi-invariant T cell receptor alpha (TCRα) chain. Inhumans, MAIT cells express high levels of CD161, IL-18 receptor, andchemokine receptors CCR5, CXCR6, and CCR6 on the cell surface. MAITcells are thought to play a role in autoimmune diseases, such asmultiple sclerosis, arthritis, and inflammatory bowel disease. Inspecific embodiments, the MAIT cells target cells infected byintracellular bacteria.

KLRB1 demonstrates increased expression in certain cells types andtissues know to function as HIV reservoirs. Bradley et al. Cell Reports,25:107-117 (2018). Accordingly in certain example embodiments, a methodof treating latent HIV infection comprises administering one or more ofthe therapeutic agents disclosed herein to a subject in need therefor.The one or more agents may be administered alone or in combination withexisting HIV therapeutic agents and anti-viral regimens. In certainexample embodiments, the one or more therapeutic agents comprises anantibody against CD161 and/or an antibody against CLEC2D.

Methods of Treating Inflammatory Diseases

In some embodiments, the method comprises treating an inflammatorydisease in a subject in need thereof by administering an agent capableof increasing KLRB1 expression or stimulating signaling through KLRB1.Inflammatory diseases include, but are not necessarily limited to,psoriasis, Crohn's disease, rheumatoid arthritis and atherosclerosis, asdescribed herein.

In some embodiments, the inflammatory disease is an autoimmune disease,and may include, but is not necessarily limited to, rheumatoidarthritis, lupus, inflammatory bowel disease, multiple sclerosis, type 1diabetes, Guillain-Barre syndrome, Grave's disease, or other autoimmunedisorder.

Methods of Screening Patients

In some embodiments, the invention provides for methods of screening oridentifying patients to be treated for cancer using the embodimentsdescribed herein. Such methods may comprise detecting the quantity ofCD161+ T cells and the quantity of tumor cells and tumor-infiltratingimmune cells that express CD161 ligands in a tumor sample from asubject.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The term “sample” used for detecting or determining the presence orlevel of at least one biomarker is typically whole blood, plasma, serum,saliva, urine, stool (e.g., feces), tears, and any other bodily fluid(e.g., as described above under the definition of “body fluids”), or atissue sample (e.g., biopsy) such as a small intestine, colon sample, orsurgical resection tissue. In certain instances, the method of thepresent invention further comprises obtaining the sample from theindividual prior to detecting or determining the presence or level of atleast one marker in the sample.

The term “immune cell” refers to cells that play a role in the immuneresponse. Immune cells are of hematopoietic origin, and includelymphocytes, such as B cells and T cells; natural killer cells; myeloidcells, such as monocytes, macrophages, eosinophils, mast cells,basophils, and granulocytes.

Immune cells can be obtained from a single source or a plurality ofsources (e.g., a single subject or a plurality of subjects). A pluralityrefers to at least two (e.g., more than one). In still anotherembodiment, the non-human mammal is a mouse. The animals from which celltypes of interest are obtained may be adult, newborn (e.g., less than 48hours old), immature, or in utero. Cell types of interest may be primarycells, stem cells, established cancer cell lines, immortalized primarycells, and the like.

The term “T cell” includes CD4⁺ T cells and CD8⁺ T cells. The term Tcell also includes both T helper 1 type T cells and T helper 2 type Tcells. The term “antigen presenting cell” includes professional antigenpresenting cells (e.g., B lymphocytes, monocytes, dendritic cells,Langerhans cells), as well as other antigen presenting cells (e.g.,keratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes).

Conventional T cells, also known as Tconv or Teffs, have effectorfunctions (e.g., cytokine secretion, cytotoxic activity,anti-self-recognization, and the like) to increase immune responses byvirtue of their expression of one or more T cell receptors. Tcons orTeffs are generally defined as any T cell population that is not a Tregand include, for example, naïve T cells, activated T cells, memory Tcells, resting Tcons, or Tcons that have differentiated toward, forexample, the Th1 or Th2 lineages. In some embodiments, Teffs are asubset of non-Treg T cells. In some embodiments, Teffs are CD4+ Teffs orCD8+ Teffs, such as CD4+ helper T lymphocytes (e.g., Th0, Th1, Tfh, orTh17) and CD8+ cytotoxic T lymphocytes. As described further herein,cytotoxic T cells are CD8+ T lymphocytes. “Naïve Tcons” are CD4⁺ T cellsthat have differentiated in bone marrow, and successfully underwent apositive and negative processes of central selection in a thymus, buthave not yet been activated by exposure to an antigen. Naïve Tcons arecommonly characterized by surface expression of L-selectin (CD62L),absence of activation markers such as CD25, CD44 or CD69, and absence ofmemory markers such as CD45RO. Naïve Tcons are therefore believed to bequiescent and non-dividing, requiring interleukin-7 (IL-7) andinterleukin-15 (IL- 15) for homeostatic survival (see, at least WO2010/101870). The presence and activity of such cells are undesired inthe context of suppressing immune responses. Unlike Tregs, Tcons are notanergic and can proliferate in response to antigen-based T cell receptoractivation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. Biol.Sci. 356:625-637). In tumors, exhausted cells can present hallmarks ofanergy.

The term “immune response” includes T cell mediated and/or B cellmediated immune responses that are influenced by modulation of T cellcostimulation. Exemplary immune responses include T cell responses,e.g., cytokine production, and cellular cytotoxicity. In addition, theterm immune response includes immune responses that are indirectlyeffected by T cell activation, e.g., antibody production (humoralresponses) and activation of cytokine responsive cells, e.g.,macrophages.

The term “CD161” or “KLRB1”, also known as killer cell lectin likereceptor B1, NKR, CD161, CLEC5B, NKR-P1, NKRP1A, NKR-P1A, hNKR-P1A,refers to a gene of the C-type lectin superfamily. The KLRB1 proteincontains an extracellular domain with several motifs characteristic ofC-type lectins, a transmembrane domain, and a cytoplasmic domain. TheKLRB1 protein is classified as a type II membrane protein because it hasan external C terminus. KLRB1 Plays an inhibitory role on natural killer(NK) cells cytotoxicity. Activation of KLRB1 results in specific acidsphingomyelinase/SMPD1 stimulation with subsequent marked elevation ofintracellular ceramide. Activation of KLRB1 also leads to AKT1/PKB andRPS6KA1/RSK1 kinases stimulation as well as markedly enhanced T-cellproliferation induced by anti-CD3. KLRB1 protein acts as a lectin thatbinds to the terminal carbohydrate Gal-alpha(1,3)Gal epitope as well asto the N-acetyllactosamine epitope. KLRB1 protein also binds toCLEC2D/LLT1 as a ligand and inhibits NK cell-mediated cytotoxicity aswell as interferon-gamma secretion in target cells.

The term “KLRB1” is intended to include fragments, variants (e.g.,allelic variants), and derivatives thereof. Representative human KLRB1cDNA and human KLRB1 protein sequences are well-known in the art and arepublicly available from the National Center for BiotechnologyInformation (NCBI). For example, at least one human KLRB1 isoform isknown: human KLRB1 (NP_002249.1) is encodable by the transcript(NM_002258.2). Nucleic acid and polypeptide sequences of KLRB1 orthologsin organisms other than humans are well-known and include, for example,chimpanzee KLRB1 (XM_001141049.2 and XP_001141049.2), Rhesus monkeyKLRB1 (XM_015151052.1 and XP_015006538.1, and XM_015151053.1 andXP_015006539.1), dog KLRB1 (XM_005637170.3 and XP_005637227.1), cattleKLRB1 (NM_001206636.1 and NP_001193565.1), mouse Klrb1 (NM_001099918.1and NP_001093388.1), and rat Klrb1 (NM_001085405.1 and NP_001078874.1).Representative sequences of KLRB1 orthologs are presented below in Table2.

In specific embodiments, the CDC161 ligand on the tumor-infiltratingimmune cells is CLEC2D.

The term “CLEC2D”, also known as C-type lectin domain family 2 member D,CLAX, LLT1, OCIL, refers to a member of the natural killer cell receptorC-type lectin family. The CLEC2D protein contains a transmembrane domainnear the N-terminus as well as the C-type lectin-like extracellulardomain. CLEC2D is a receptor for KLRB1 that protects target cellsagainst natural killer cell-mediated lysis. CLEC2D inhibits osteoclastformation and bone resorption. CLEC2D also modulates the release ofinterferon-gamma, and binds high molecular weight sulfatedglycosaminoglycans. Diseases associated with CLEC2D include inflammatorybowel disease. Among its related pathways are Class I MHC mediatedantigen processing and presentation and Innate Immune System.

The term “CLEC2D” is intended to include fragments, variants (e.g.,allelic variants), and derivatives thereof Representative human CLEC2DcDNA and human CLEC2D protein sequences are well-known in the art andare publicly available from the National Center for BiotechnologyInformation (NCBI). Human CLEC2D isoforms include isoform 1 (NM_013269.5and NP_037401.1), isoform 2 (NM_001004419.4 and NP_001004419.1; whichincludes an additional exon, compared to variant 1, that causes aframeshift, resulting a longer isoform with a distinct C-terminuscompared to isoform 1), isoform 3 (NM_001197317.2 and NP_001184246.1;which lacks an in-frame exon in the 5′ region, resulting an isoform thatlacks an internal segment compared to isoform 1), isoform 4(NM_001197318.2 and NP_001184247.1; which lacks an exon in the 3′ regionthat causes a frame-shift, resulting a shorter isoform with a distinctC-terminus compared to isoform 1), and isoform 5 (NM_001197319.2 andNP_001184248.1; which lacks an in-frame exon in the 5′ region and anexon in the 3′ region that causes a frame-shift, resulting an isoformthat lacks an internal segment and has a shorter and distinct C-terminuscompared to isoform 1). Nucleic acid and polypeptide sequences of CLEC2Dorthologs in organisms other than humans are well-known and include, forexample, chimpanzee CLEC2D (XM_016922954.1 and XP_016778443.1,XM_016922955.1 and XP_016778444.1, XM_001142174.5 and XP_001142174.1,and XM_016922953.1 and XP_016778442.1), Rhesus monkey CLEC2D(XM_015151056.1 and XP_015006542.1, XM_015151055.1 and XP_015006541.1,and XM_015151057.1 and XP_015006543.1), dog CLEC2D (XM_022411270.1 andXP_022266978.1, XM_022411269.1 and XP_022266977.1, XM_005637169.3 andXP_005637226.1, and XM_022411268.1 and XP_022266976.1), cattle CLEC2D(XM_002687838.5 and XP_002687884.1, and XM_015471294.1 andXP_015326780.1), mouse CLEC2D (NM_053109.3 and NP_444339.1), and ratCLEC2D (NM_130402.2 and NP_569086.1). Representative sequences of CLEC2Dorthologs are presented below in Table 2.

Methods of Selecting Agents that Modulate Immune Cell Activation

Another aspect of the present invention relates to methods of selectingagents (e.g., antibodies, fusion proteins, peptides, or small molecules)which modulate an immune response by modulating costimulation, such asagents that inhibit or promote the interaction between KLRB1 and one ormore natural binding partners, such as CLEC2D. Such methods utilizescreening assays, including cell based and non-cell based assays. In oneembodiment, the assays provide a method for identifying agents thatinhibit the interaction between KLRB1 and one or more natural bindingpartners, such as CLEC2D.

Certain definitions are useful for describing this and other aspects ofthe present invention.

The term “activity,” when used with respect to a polypeptide, e.g.,KLRB1 and/or a KLRB1 natural binding partner, such as the ligand,CLEC2D, includes activities that are inherent in the structure of theprotein. For example, with regard to a KLRB1 ligand, the term “activity”includes the ability to modulate immune cell inhibition by modulating aninhibitory signal in an immune cell (e.g., by engaging a naturalreceptor on an immune cell). Those of skill in the art will recognizethat when an activating form of the KLRB1 ligand polypeptide binds to aninhibitory receptor, an inhibitory signal is generated in the immunecell.

The term “altered amount” or “altered level” refers to increased ordecreased copy number (e.g., germline and/or somatic) of a biomarkernucleic acid, e.g., increased or decreased expression level in a cancersample, as compared to the expression level or copy number of thebiomarker nucleic acid in a control sample. The term “altered amount” ofa biomarker also includes an increased or decreased protein level of abiomarker protein in a sample, e.g., a cancer sample, as compared to thecorresponding protein level in a normal, control sample. Furthermore, analtered amount of a biomarker protein may be determined by detectingposttranslational modification such as methylation status of the marker,which may affect the expression or activity of the biomarker protein.

The amount of a biomarker in a subject is “significantly” higher orlower than the normal amount of the biomarker, if the amount of thebiomarker is greater or less, respectively, than the normal or controllevel by an amount greater than the standard error of the assay employedto assess amount, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%,900%, 1000% or than that amount. Alternatively, the amount of thebiomarker in the subject can be considered “significantly” higher orlower than the normal and/or control amount if the amount is at leastabout two, and preferably at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%,45%, 4 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%,165%, 170%, 175%, 180%, 185%, 190%, 195%, two times, three times, fourtimes, five times, or more, or any range in between, such as 5%-100%,higher or lower, respectively, than the normal and/or control amount ofthe biomarker. Such significant modulation values can be applied to anymetric described herein, such as altered level of expression, alteredactivity, changes in cancer cell hyperproliferative growth, changes incancer cell death, changes in biomarker inhibition, changes in testagent binding, and the like.

The term “altered level of expression” of a marker refers to anexpression level or copy number of a marker in a test sample e.g., asample derived from a subject suffering from cancer, that is greater orless than the standard error of the assay employed to assess expressionor copy number, and is preferably at least twice, and more preferablythree, four, five or ten or more times the expression level or copynumber of the marker or chromosomal region in a control sample (e.g.,sample from a healthy subject not having the associated disease) andpreferably, the average expression level or copy number of the marker orchromosomal region in several control samples. The altered level ofexpression is greater or less than the standard error of the assayemployed to assess expression or copy number, and is preferably at leasttwice, and more preferably three, four, five or ten or more times theexpression level or copy number of the marker in a control sample (e.g.,sample from a healthy subject not having the associated disease) andpreferably, the average expression level or copy number of the marker inseveral control samples.

The term “altered activity” of a marker refers to an activity of amarker which is increased or decreased in a disease state, e.g., in acancer sample, as compared to the activity of the marker in a normal,control sample. Altered activity of a marker may be the result of, forexample, altered expression of the marker, altered protein level of themarker, altered structure of the marker, or, e.g., an alteredinteraction with other proteins involved in the same or differentpathway as the marker, or altered interaction with transcriptionalactivators or inhibitors.

The term “altered structure” of a biomarker refers to the presence ofmutations or allelic variants within a biomarker nucleic acid orprotein, e.g., mutations which affect expression or activity of thebiomarker nucleic acid or protein, as compared to the normal orwild-type gene or protein. For example, mutations include, but are notlimited to substitutions, deletions, or addition mutations. Mutationsmay be present in the coding or non-coding region of the biomarkernucleic acid.

The “amount” of a marker, e.g., expression or copy number of a marker orMCR, or protein level of a marker, in a subject is “significantly”higher or lower than the normal amount of a marker, if the amount of themarker is greater or less, respectively, than the normal level by anamount greater than the standard error of the assay employed to assessamount, and preferably at least twice, and more preferably three, four,five, ten or more times that amount. Alternately, the amount of themarker in the subject can be considered “significantly” higher or lowerthan the normal amount if the amount is at least about two, andpreferably at least about three, four, or five times, higher or lower,respectively, than the normal amount of the marker.

The term “body fluid” refers to fluids that are excreted or secretedfrom the body as well as fluids that are normally not (e.g. amnioticfluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid,cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle,chyme, stool, female ejaculate, interstitial fluid, intracellular fluid,lymph, menses, breast milk, mucus, pleural fluid, peritoneal fluid, pus,saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine,vaginal lubrication, vitreous humor, vomit).

The term “control” refers to any reference standard suitable to providea comparison to the expression products in the test sample. In oneembodiment, the control comprises obtaining a “control sample” fromwhich expression product levels are detected and compared to theexpression product levels from the test sample. Such a control samplemay comprise any suitable sample, including but not limited to a samplefrom a control patient (can be stored sample or previous samplemeasurement) with a known outcome; normal tissue or cells isolated froma subject, such as a normal patient or the patient having a condition ofinterest (cancer is used below as a representative condition), culturedprimary cells/tissues isolated from a subject such as a normal subjector the cancer patient, adjacent normal cells/tissues obtained from thesame organ or body location of the cancer patient, a tissue or cellsample isolated from a normal subject, or a primary cells/tissuesobtained from a depository. In another preferred embodiment, the controlmay comprise a reference standard expression product level from anysuitable source, including but not limited to housekeeping genes, anexpression product level range from normal tissue (or other previouslyanalyzed control sample), a previously determined expression productlevel range within a test sample from a group of patients, or a set ofpatients with a certain outcome (for example, survival for one, two,three, four years, etc.) or receiving a certain treatment (for example,standard of care cancer therapy). It will be understood by those ofskill in the art that such control samples and reference standardexpression product levels can be used in combination as controls in themethods of the present invention. In one embodiment, the control maycomprise normal or non-cancerous cell/tissue sample. In anotherpreferred embodiment, the control may comprise an expression level for aset of patients, such as a set of cancer patients, or for a set ofcancer patients receiving a certain treatment, or for a set of patientswith one outcome versus another outcome. In the former case, thespecific expression product level of each patient can be assigned to apercentile level of expression, or expressed as either higher or lowerthan the mean or average of the reference standard expression level. Inanother preferred embodiment, the control may comprise normal cells,cells from patients treated with combination chemotherapy, and cellsfrom patients having benign cancer. In another embodiment, the controlmay also comprise a measured value for example, average level ofexpression of a particular gene in a population compared to the level ofexpression of a housekeeping gene in the same population. Such apopulation may comprise normal subjects, cancer patients who have notundergone any treatment (i.e., treatment naive), cancer patientsundergoing standard of care therapy, or patients having benign cancer.In another preferred embodiment, the control comprises a ratiotransformation of expression product levels, including but not limitedto determining a ratio of expression product levels of two genes in thetest sample and comparing it to any suitable ratio of the same two genesin a reference standard; determining expression product levels of thetwo or more genes in the test sample and determining a difference inexpression product levels in any suitable control; and determiningexpression product levels of the two or more genes in the test sample,normalizing their expression to expression of housekeeping genes in thetest sample, and comparing to any suitable control. In particularlypreferred embodiments, the control comprises a control sample which isof the same lineage and/or type as the test sample. In anotherembodiment, the control may comprise expression product levels groupedas percentiles within or based on a set of patient samples, such as allpatients with cancer. In one embodiment a control expression productlevel is established wherein higher or lower levels of expressionproduct relative to, for instance, a particular percentile, are used asthe basis for predicting outcome. In another preferred embodiment, acontrol expression product level is established using expression productlevels from cancer control patients with a known outcome, and theexpression product levels from the test sample are compared to thecontrol expression product level as the basis for predicting outcome. Asdemonstrated by the data below, the methods of the present invention arenot limited to use of a specific cut-point in comparing the level ofexpression product in the test sample to the control.

The “copy number” of a biomarker nucleic acid refers to the number ofDNA sequences in a cell (e.g., germline and/or somatic) encoding aparticular gene product. Generally, for a given gene, a mammal has twocopies of each gene. The copy number can be increased, however, by geneamplification or duplication, or reduced by deletion. For example,germline copy number changes include changes at one or more genomicloci, wherein said one or more genomic loci are not accounted for by thenumber of copies in the normal complement of germline copies in acontrol (e.g., the normal copy number in germline DNA for the samespecies as that from which the specific germline DNA and correspondingcopy number were determined). Somatic copy number changes includechanges at one or more genomic loci, wherein said one or more genomicloci are not accounted for by the number of copies in germline DNA of acontrol (e.g., copy number in germline DNA for the same subject as thatfrom which the somatic DNA and corresponding copy number weredetermined).

The “normal” copy number (e.g., germline and/or somatic) of a biomarkernucleic acid or “normal” level of expression of a biomarker nucleicacid, or protein is the activity/level of expression or copy number in abiological sample, e.g., a sample containing tissue, whole blood, serum,plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, andbone marrow, from a subject, e.g., a human, not afflicted with cancer,or from a corresponding non-cancerous tissue in the same subject who hascancer.

A molecule is “fixed” or “affixed” to a substrate if it is covalently ornon-covalently associated with the substrate such the substrate can berinsed with a fluid (e.g. standard saline citrate, pH 7.4) without asubstantial fraction of the molecule dissociating from the substrate.

The terms “high,” “low,” “intermediate,” and “negative” in connectionwith cellular biomarker expression refers to the amount of the biomarkerexpressed relative to the cellular expression of the biomarker by one ormore reference cells. Biomarker expression can be determined accordingto any method described herein including, without limitation, ananalysis of the cellular level, activity, structure, and the like, ofone or more biomarker genomic nucleic acids, ribonucleic acids, and/orpolypeptides. In one embodiment, the terms refer to a defined percentageof a population of cells expressing the biomarker at the highest,intermediate, or lowest levels, respectively. Such percentages can bedefined as the top 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%,4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10%,11%, 12%, 13%, 14%, 15% or more, or any range in between, inclusive, ofa population of cells that either highly express or weakly express thebiomarker. The term “low” excludes cells that do not detectably expressthe biomarker, since such cells are “negative” for biomarker expression.The term “intermediate” includes cells that express the biomarker, butat levels lower than the population expressing it at the “high” level.In another embodiment, the terms can also refer to, or in thealternative refer to, cell populations of biomarker expressionidentified by qualitative or statistical plot regions. For example, cellpopulations sorted using flow cytometry can be discriminated on thebasis of biomarker expression level by identifying distinct plots basedon detectable moiety analysis, such as based on mean fluorescenceintensities and the like, according to well-known methods in the art.Such plot regions can be refined according to number, shape, overlap,and the like based on well-known methods in the art for the biomarker ofinterest. In still another embodiment, the terms can also be determinedaccording to the presence or absence of expression for additionalbiomarkers.

The term “immunotherapeutic agent” can include any molecule, peptide,antibody or other agent which can stimulate a host immune system togenerate an immune response to a tumor or cancer in the subject. Variousimmunotherapeutic agents are useful in the compositions and methodsdescribed herein.

The term “inhibit” or “downregulate” includes the decrease, limitation,or blockage, of, for example a particular action, function, orinteraction. In some embodiments, cancer is “inhibited” if at least onesymptom of the cancer is alleviated, terminated, slowed, or prevented.As used herein, cancer is also “inhibited” if recurrence or metastasisof the cancer is reduced, slowed, delayed, or prevented. Similarly, abiological function, such as the function of a protein, is inhibited ifit is decreased as compared to a reference state, such as a control likea wild-type state. For example, binding of KLRB1 and CLEC2D is inhibitedby an agent if the agent reduces the physical interaction of interestbetween KLRB1 and CLEC2D, such as KLRB1 expressed by a T cell. Suchinhibition or deficiency can be induced, such as by application of agentat a particular time and/or place, or can be constitutive, such as by aheritable mutation. Such inhibition or deficiency can also be partial orcomplete (e.g., essentially no measurable activity in comparison to areference state, such as a control like a wild-type state). Essentiallycomplete inhibition or deficiency is referred to as blocked. The term“promote” or “upregulate” has the opposite meaning.

The term “inhibitory signal” refers to a signal transmitted via aninhibitory receptor (e.g., KLRB1, CTLA4, PD-1, and the like) for apolypeptide on a immune cell. Such a signal antagonizes a signal via anactivating receptor (e.g., via a TCR, CD3, BCR, or Fc polypeptide) andcan result in, e.g., inhibition of second messenger generation; aninhibition of proliferation; an inhibition of effector function in theimmune cell, e.g., reduced phagocytosis, reduced antibody production,reduced cellular cytotoxicity, the failure of the immune cell to producemediators, (such as cytokines (e.g., IL-2) and/or mediators of allergicresponses); or the development of anergy.

The term “interaction,” when referring to an interaction between twomolecules, refers to the physical contact (e.g., binding) of themolecules with one another. Generally, such an interaction results in anactivity (which produces a biological effect) of one or both of saidmolecules. The activity may be a direct activity of one or both of themolecules, (e.g., signal transduction). Alternatively, one or bothmolecules in the interaction may be prevented from binding their ligand,and thus be held inactive with respect to ligand binding activity (e.g.,binding its ligand and triggering or inhibiting costimulation). Toinhibit such an interaction results in the disruption of the activity ofone or more molecules involved in the interaction. To enhance such aninteraction is to prolong or increase the likelihood of said physicalcontact, and prolong or increase the likelihood of said activity.

The term “K_(D)” is intended to refer to the dissociation equilibriumconstant of a particular antibody-antigen interaction. The bindingaffinity of antibodies of the disclosed invention may be measured ordetermined by standard antibody-antigen assays, for example, competitiveassays, saturation assays, or standard immunoassays such as ELISA orRIA.

The term “modulate” includes up-regulation and down-regulation, e.g.,enhancing or inhibiting a response.

The “normal” level of expression of a marker is the level of expressionof the marker in cells of a subject, e.g., a human patient, notafflicted with a cancer. An “over-expression” or “significantly higherlevel of expression” of a marker refers to an expression level in a testsample that is greater than the standard error of the assay employed toassess expression, and is preferably at least twice, and more preferably2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 times or more higher than the expression activity or level of themarker in a control sample (e.g., sample from a healthy subject nothaving the marker associated disease) and preferably, the averageexpression level of the marker in several control samples. A“significantly lower level of expression” of a marker refers to anexpression level in a test sample that is at least twice, and morepreferably 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 times or more lower than the expression level of themarker in a control sample (e.g., sample from a healthy subject nothaving the marker associated disease) and preferably, the averageexpression level of the marker in several control samples. An“over-expression” or “significantly higher level of expression” of abiomarker refers to an expression level in a test sample that is greaterthan the standard error of the assay employed to assess expression, andis preferably at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 times or more higher than the expressionactivity or level of the biomarker in a control sample (e.g., samplefrom a healthy subject not having the biomarker associated disease) andpreferably, the average expression level of the biomarker in severalcontrol samples. A “significantly lower level of expression” of abiomarker refers to an expression level in a test sample that is atleast 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 times or more lower than the expression level of thebiomarker in a control sample (e.g., sample from a healthy subject nothaving the biomarker associated disease) and preferably, the averageexpression level of the biomarker in several control samples. An“underexpression” or “significantly lower level of expression or copynumber” of a marker (e.g., KLRB1 and/or a natural binding partnerthereof, such as CLEC2D, and/or downstream signaling marker thereof)refers to an expression level or copy number in a test sample that isgreater than the standard error of the assay employed to assessexpression or copy number, but is preferably at least twice, and morepreferably three, four, five or ten or more times less than theexpression level or copy number of the marker in a control sample (e.g.,sample from a healthy subject not afflicted with cancer) and preferably,the average expression level or copy number of the marker in severalcontrol samples.

Such “significance” levels can also be applied to any other measuredparameter described herein, such as for expression, inhibition,cytotoxicity, cell growth, and the like.

The term “pre-determined” biomarker amount and/or activitymeasurement(s) may be a biomarker amount and/or activity measurement(s)used to, by way of example only, evaluate a subject that may be selectedfor a particular treatment, evaluate a response to a treatment such asone or more modulators of the KLRB1 pathway, such as a modulator ofKLRB1 and one or more natural binding partners, such as CLEC2D, eitheralone or in combination with one or more immunotherapies, and/orevaluate the disease state. A pre-determined biomarker amount and/oractivity measurement(s) may be determined in populations of patientswith or without cancer. The pre-determined biomarker amount and/oractivity measurement(s) can be a single number, equally applicable toevery patient, or the pre-determined biomarker amount and/or activitymeasurement(s) can vary according to specific subpopulations ofpatients. Age, weight, height, and other factors of a subject may affectthe pre-determined biomarker amount and/or activity measurement(s) ofthe individual. Furthermore, the pre-determined biomarker amount and/oractivity can be determined for each subject individually. In oneembodiment, the amounts determined and/or compared in a method describedherein are based on absolute measurements. In another embodiment, theamounts determined and/or compared in a method described herein arebased on relative measurements, such as ratios (e.g., cell ratios orserum biomarker normalized to the expression of housekeeping orotherwise generally constant biomarker). The pre-determined biomarkeramount and/or activity measurement(s) can be any suitable standard. Forexample, the pre-determined biomarker amount and/or activitymeasurement(s) can be obtained from the same or a different human forwhom a patient selection is being assessed. In one embodiment, thepre-determined biomarker amount and/or activity measurement(s) can beobtained from a previous assessment of the same patient. In such amanner, the progress of the selection of the patient can be monitoredover time. In addition, the control can be obtained from an assessmentof another human or multiple humans, e.g., selected groups of humans, ifthe subject is a human. In such a manner, the extent of the selection ofthe human for whom selection is being assessed can be compared tosuitable other humans, e.g., other humans who are in a similar situationto the human of interest, such as those suffering from similar or thesame condition(s) and/or of the same ethnic group.

The term “predictive” includes the use of a biomarker nucleic acidand/or protein status, e.g., over- or under- activity, emergence,expression, growth, remission, recurrence or resistance of tumorsbefore, during or after therapy, for determining the likelihood ofresponse of a cancer to immunomodulatory therapy, such as KLRB1 pathwaymodulator therapy (e.g., modulator of the interaction between KLRB1 andone or more natural binding partners, such as CLEC2D, either alone or incombination with an immunotherapy, such as an immune checkpointinhibition therapy). Such predictive use of the biomarker may beconfirmed by, e.g., (1) increased or decreased copy number (e.g., byFISH, FISH plus SKY, single-molecule sequencing, e.g., as described inthe art at least at J. Biotechnol., 86:289-301, or qPCR), overexpressionor underexpression of a biomarker nucleic acid (e.g., by ISH, NorthernBlot, or qPCR), increased or decreased biomarker protein (e.g., by IHC)and/or biomarker target, or increased or decreased activity, e.g., inmore than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%,25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of assayedhuman cancers types or cancer samples; (2) its absolute or relativelymodulated presence or absence in a biological sample, e.g., a samplecontaining tissue, whole blood, serum, plasma, buccal scrape, saliva,cerebrospinal fluid, urine, stool, or bone marrow, from a subject, e.g.a human, afflicted with cancer; (3) its absolute or relatively modulatedpresence or absence in clinical subset of patients with cancer (e.g.,those responding to a particular immunomodulatory therapy (e.g., KLRB1pathway modulator therapy (e.g., modulator of the interaction betweenKLRB1 and one or more natural binding partners, such as CLEC2D, eitheralone or in combination with an immunotherapy) or those developingresistance thereto).

The term “response to therapy” (e.g., KLRB1 pathway modulator therapy(e.g., modulator of the interaction between KLRB1 and one or morenatural binding partners, such as CLEC2D, either alone or in combinationwith an immunotherapy, such as an immune checkpoint inhibition therapy)relates to any response to therapy (e.g., KLRB1 pathway modulatortherapy (e.g., modulator of the interaction between KLRB1 and one ormore natural binding partners, such as CLEC2D, either alone or incombination with an immunotherapy, such as an immune checkpointinhibition therapy), and, for cancer, preferably to a change in cancercell numbers, tumor mass, and/or volume after initiation of neoadjuvantor adjuvant chemotherapy. Hyperproliferative disorder response may beassessed, for example for efficacy or in a neoadjuvant or adjuvantsituation, where the size of a tumor after systemic intervention can becompared to the initial size and dimensions as measured by CT, PET,mammogram, ultrasound or palpation. Responses may also be assessed bycaliper measurement or pathological examination of the tumor afterbiopsy or surgical resection. Response may be recorded in a quantitativefashion like percentage change in tumor volume or in a qualitativefashion like “pathological complete response” (pCR), “clinical completeremission” (cCR), “clinical partial remission” (cPR), “clinical stabledisease” (cSD), “clinical progressive disease” (cPD) or otherqualitative criteria. Assessment of hyperproliferative disorder responsemay be done early after the onset of neoadjuvant or adjuvant therapy,e.g., after a few hours, days, weeks or preferably after a few months. Atypical endpoint for response assessment is upon termination ofneoadjuvant chemotherapy or upon surgical removal of residual tumorcells and/or the tumor bed. This is typically three months afterinitiation of neoadjuvant therapy. In some embodiments, clinicalefficacy of the therapeutic treatments described herein may bedetermined by measuring the clinical benefit rate (CBR). The clinicalbenefit rate is measured by determining the sum of the percentage ofpatients who are in complete remission (CR), the number of patients whoare in partial remission (PR) and the number of patients having stabledisease (SD) at a time point at least 6 months out from the end oftherapy. The shorthand for this formula is CBR=CR+PR+SD over 6 months.In some embodiments, the CBR for a particular cancer therapeutic regimenis at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, or more. Additional criteria for evaluating the response to cancertherapies are related to “survival,” which includes all of thefollowing: survival until mortality, also known as overall survival(wherein said mortality may be either irrespective of cause or tumorrelated); “recurrence-free survival” (wherein the term recurrence shallinclude both localized and distant recurrence); metastasis freesurvival; disease free survival (wherein the term disease shall includecancer and diseases associated therewith). The length of said survivalmay be calculated by reference to a defined start point (e.g., time ofdiagnosis or start of treatment) and end point (e.g., death, recurrenceor metastasis). In addition, criteria for efficacy of treatment can beexpanded to include response to chemotherapy, probability of survival,probability of metastasis within a given time period, and probability oftumor recurrence. For example, in order to determine appropriatethreshold values, a particular cancer therapeutic regimen can beadministered to a population of subjects and the outcome can becorrelated to biomarker measurements that were determined prior toadministration of any immunomodulatory therapy. The outcome measurementmay be pathologic response to therapy given in the neoadjuvant setting.Alternatively, outcome measures, such as overall survival anddisease-free survival can be monitored over a period of time forsubjects following immunomodulatory therapy for whom biomarkermeasurement values are known. In certain embodiments, the dosesadministered are standard doses known in the art for cancer therapeuticagents. The period of time for which subjects are monitored can vary.For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months.

The terms “response” or “responsiveness” refers to response to therapy.For example, an anti-cancer response includes reduction of tumor size orinhibiting tumor growth. The terms can also refer to an improvedprognosis, for example, as reflected by an increased time to recurrence,which is the period to first recurrence censoring for second primarycancer as a first event or death without evidence of recurrence, or anincreased overall survival, which is the period from treatment to deathfrom any cause. To respond or to have a response means there is abeneficial endpoint attained when exposed to a stimulus. Alternatively,a negative or detrimental symptom is minimized, mitigated or attenuatedon exposure to a stimulus. It will be appreciated that evaluating thelikelihood that a tumor or subject will exhibit a favorable response isequivalent to evaluating the likelihood that the tumor or subject willnot exhibit favorable response (i.e., will exhibit a lack of response orbe non-responsive).

The term “KLRB1 pathway” includes KLRB1 and interactions of KLRB1 withone or more of its natural binding partners, such as CLEC2D.

In one embodiment, the invention relates to assays for screeningcandidate or test compounds that bind to, or modulate the activity of,KLRB1 and/or one or more natural binding partners, such as CLEC2D. Inone embodiment, a method for identifying an agent to modulate an immuneresponse entails determining the ability of the agent to modulate, e.g.enhance or inhibit, the interaction between KLRB1 and one or morenatural binding partners, such as CLEC2D. In one embodiment, an agentthat modulates the interaction between KLRB1 and one or more naturalbinding partners, such as CLEC2D, is selected. Such agents include,without limitation, antibodies, proteins, fusion proteins, smallmolecules, and nucleic acids.

In one embodiment, a method for identifying an agent which enhances animmune response entails determining the ability of the candidate agentto enhance the interaction between KLRB1 and one or more natural bindingpartners, such as CLEC2D.

In another embodiment, a method for identifying an agent to upregulatean immune response entails determining the ability of a candidate agentto inhibit the interaction between KLRB1 and one or more natural bindingpartners, such as CLEC2D. In another embodiment, a method foridentifying an agent to downregulate an immune response entailsdetermining the ability of the candidate agent to enhance theinteraction between KLRB1 and one or more natural binding partners, suchas CLEC2D.

In one embodiment, an assay is a cell-based assay, comprising contacting(a) a cell expressing KLRB1 with one or more KLRB1 natural bindingpartners, such as CLEC2D or (b) a cell expressing one or more KLRB1natural binding partners, such as CLEC2D, with KLRB1, with a testcompound and determining the ability of the test compound to modulate(e.g. stimulate or inhibit) the binding between KLRB1 and the one ormore natural binding partners, such as CLEC2D. Determining the abilityof the polypeptides to bind to, or interact with, each other can beaccomplished, e.g., by measuring direct binding or by measuring aparameter of immune cell activation.

For example, in a direct binding assay, the polypeptides can be coupledwith a radioisotope or enzymatic label such that binding of KLRB1 andone or more natural binding partners, such as CLEC2D can be determinedby detecting the labeled protein in a complex. For example, thepolypeptides can be labeled with ¹²I, ³⁵S, ¹⁴C, or ³H, either directlyor indirectly, and the radioisotope detected by direct counting ofradioemmission or by scintillation counting. Alternatively, thepolypeptides can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

It is also within the scope of the present invention to determine theability of a compound to modulate the interaction between KLRB1 and oneor more natural binding partners, such as CLEC2D, without the labelingof any of the interactants. For example, a microphysiometer can be usedto detect the interaction between KLRB1 and one or more natural bindingpartners, such as CLEC2D without the labeling of either polypeptide(McConnell, H. M. et al. (1992) Science 257:1906-1912). As used herein,a “microphysiometer” (e.g., Cytosensor) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween compound and receptor.

In a preferred embodiment, determining the ability of the blockingagents (e.g. antibodies, fusion proteins, peptides, or small molecules)to antagonize the interaction between a given set of polypeptides can beaccomplished by determining the activity of one or more members of theset of polypeptides. For example, the activity of KLRB1 and/or one ormore natural binding partners, such as CLEC2D, can be determined bydetecting induction of a cellular second messenger (e.g., intracellularsignaling), detecting catalytic/enzymatic activity of an appropriatesubstrate, detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., chloramphenicol acetyltransferase), or detecting a cellular response regulated by KLRB1 and/orthe one or more natural binding partners, such as CLEC2D. Determiningthe ability of the blocking agent to bind to or interact with saidpolypeptide can be accomplished, for example, by measuring the abilityof a compound to modulate immune cell costimulation or inhibition in aproliferation assay, or by interfering with the ability of saidpolypeptide to bind to antibodies that recognize a portion thereof.

Agents that enhance interactions between KLRB1 and/or one or morenatural binding partners, such as CLEC2D, can be identified by theirability to inhibit immune cell proliferation, and/or effector function,or to induce anergy, clonal deletion, and/or exhaustion when added to anin vitro assay. For example, cells can be cultured in the presence of anagent that stimulates signal transduction via an activating receptor. Anumber of recognized readouts of cell activation can be employed tomeasure, cell proliferation or effector function (e.g., antibodyproduction, cytokine production, phagocytosis) in the presence of theactivating agent. The ability of a test agent to block this activationcan be readily determined by measuring the ability of the agent toeffect a decrease in proliferation or effector function being measured,using techniques known in the art.

For example, agents according to the present invention can be tested forthe ability to inhibit or enhance costimulation in a T cell assay, asdescribed in Freeman et al. (2000) J. Exp. Med. 192:1027 and Latchman etal. (2001) Nat. Immunol. 2:261. CD4+ T cells can be isolated from humanPBMCs and stimulated with activating anti-CD3 antibody. Proliferation ofT cells can be measured by ³H thymidine incorporation. An assay can beperformed with or without CD28 costimulation in the assay. Similarassays can be performed with Jurkat T cells and PHA-blasts from PBMCs.

Alternatively, agents of the present invention can be tested for theability to modulate cellular production of cytokines which are producedby or whose production is enhanced or inhibited in immune cells inresponse to modulation of KLRB1 and/or one or more natural bindingpartners, such as CLEC2D. For example, immune cells expressing KLRB1 canbe suboptimally stimulated in vitro with a primary activation signal,for example, T cells can be stimulated with phorbol ester, anti-CD3antibody or preferably antigen in association with an MHC class IImolecule, and given a costimulatory signal, e.g., by a stimulatory formof B7 family antigen, for instance by a cell transfected with nucleicacid encoding a B7 polypeptide and expressing the peptide on its surfaceor by a soluble, stimulatory form of the peptide. Known cytokinesreleased into the media can be identified by ELISA or by the ability ofan antibody which blocks the cytokine to inhibit immune cellproliferation or proliferation of other cell types that is induced bythe cytokine. For example, an IL-4 ELISA kit is available from Genzyme(Cambridge Mass.), as is an IL-7 blocking antibody. Blocking antibodiesagainst IL-9 and IL-12 are available from Genetics Institute (Cambridge,Mass.). The effect of stimulating or blocking the interaction of KLRB1and one or more natural binding partners, such as CLEC2D, on thecytokine profile can then be determined. An in vitro immune cellcostimulation assay as described above can also be used in a method foridentifying cytokines which can be modulated by modulation of theinteraction between KLRB1 and/or one or more natural binding partners,such as CLEC2D activity. For example, if a particular activity inducedupon costimulation, e.g., immune cell proliferation, cannot be inhibitedby addition of blocking antibodies to known cytokines, the activity mayresult from the action of an unknown cytokine. Following costimulation,this cytokine can be purified from the media by conventional methods andits activity measured by its ability to induce immune cellproliferation. To identify cytokines which may play a role the inductionof tolerance, an in vitro T cell costimulation assay as described abovecan be used. In this case, T cells would be given the primary activationsignal and contacted with a selected cytokine, but would not be giventhe costimulatory signal. After washing and resting the immune cells,the cells would be rechallenged with both a primary activation signaland a costimulatory signal. If the immune cells do not respond (e.g.,proliferate or produce cytokines) they have become tolerized and thecytokine has not prevented the induction of tolerance. However, if theimmune cells respond, induction of tolerance has been prevented by thecytokine. Those cytokines which are capable of preventing the inductionof tolerance can be targeted for blockage in vivo in conjunction withreagents which block B lymphocyte antigens as a more efficient means toinduce tolerance in transplant recipients or subjects with autoimmunediseases.

In yet another embodiment, an assay of the present invention is acell-free assay for screening for compounds which modulate theinteraction between KLRB1 and/or one or more natural binding partners,such as CLEC2D, comprising contacting a polypeptide of KLRB1 and/or oneor more natural binding partners, such as CLEC2D protein, orbiologically active portion thereof, with a test compound anddetermining the ability of the test compound to modulate the interactionbetween the KLRB1 and/or one or more natural binding partners, such asCLEC2D, or biologically active portion thereof. Binding of the testcompound can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting thepolypeptide, or biologically active portion thereof, with its bindingpartner to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with the polypeptide in the assay mixture, wherein determiningthe ability of the test compound to interact with the polypeptidecomprises determining the ability of the test compound to preferentiallybind to the polypeptide or biologically active portion thereof, ascompared to the binding partner.

For example, KLRB1 and/or one or more natural binding partners, such asCLEC2D, can be used to form an assay mixture and the ability of apolypeptide to block this interaction can be tested by determining theability of KLRB1 to bind the one or more natural binding partners, suchas CLEC2D, by one of the methods described above for determining directbinding. In some embodiments, whether for cell-based or cell-freeassays, the test compound can further be assayed to determine whether itaffects binding and/or activity of the interaction between KLRB1 and/orthe one or more natural binding partners, such as CLEC2D, with otherbinding partners. Other useful binding analysis methods include the useof real-time Biomolecular Interaction Analysis (BIA) (Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” is atechnology for studying biospecific interactions in real time, withoutlabeling any of the interactants (e.g., BIAcore). Changes in the opticalphenomenon of surface plasmon resonance (SPR) can be used as anindication of real-time reactions between biological polypeptides.Polypeptides of interest can be immobilized on a BIAcore chip andmultiple agents (blocking antibodies, fusion proteins, peptides, orsmall molecules) can be tested for binding to the polypeptide ofinterest. An example of using the BIA technology is described by Fitz etal. (1997) Oncogene 15:613.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of proteins. In the case ofcell-free assays in which a membrane-bound form protein is used (e.g., acell surface KLRB1 and/or one or more natural binding partners, such asCLEC2D) it may be desirable to utilize a solubilizing agent such thatthe membrane-bound form of the protein is maintained in solution.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In one or more embodiments of the above described assay methods, it maybe desirable to immobilize either polypeptides to facilitate separationof complexed from uncomplexed forms of one or both of the proteins, aswell as to accommodate automation of the assay. Binding of a testcompound to a polypeptide, can be accomplished in any vessel suitablefor containing the reactants. Examples of such vessels includemicrotiter plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-S-transferase-based polypeptide fusion proteins, orglutathione-S-transferase/target fusion proteins, can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound, and the mixture incubated under conditions conduciveto complex formation (e.g., at physiological conditions for salt andpH). Following incubation, the beads or microtiter plate wells arewashed to remove any unbound components, the matrix immobilized in thecase of beads, complex determined either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of polypeptide binding oractivity determined using standard techniques.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a polypeptide of interest (e.g.,KLRB1 and/or one or more natural binding partners, such as CLEC2D) canbe accomplished as described above for cell-based assays, such as bydetermining the ability of the test compound to modulate the activity ofa polypeptide that functions downstream of the polypeptide. For example,levels of second messengers can be determined, the activity of theinteractor polypeptide on an appropriate target can be determined, orthe binding of the interactor to an appropriate target can be determinedas previously described.

The present invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthe present invention to further use an agent identified as describedherein in an appropriate animal model. For example, an agent identifiedas described herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, the present invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

Further embodiments of the invention are described in the followingnumbered paragraphs.

1. A method of treating cancer in a subject in need thereof comprisingadministering an agent capable of blocking the interaction of KLRB1 withits ligand.2. The method according to paragraph 1, wherein the KLRB1 ligand isCLEC2D.3. The method according to paragraph 1 or 2, wherein the agent comprisesan antibody or fragment thereof.4. The method according to paragraph 3, wherein the antibody is ahumanized or chimeric antibody.5. The method according to paragraph 3 or 4, wherein the antibody bindsKLRB1.6. The method according to paragraph 3 or 4, wherein the antibody bindsCLEC2D.7. The method according to paragraph 1 or 2, wherein the agent is asoluble KLRB1 protein or fragment thereof.8. The method according to paragraph 1 or 2, wherein the agent is aprogrammable nucleic acid modifying agent.9. The method according to paragraph 8, wherein the programmable nucleicacid modifying agent is a CRISPR-Cas system, a zinc finger system, aTALE system, or a meganuclease.10. The method according to paragraph 9, wherein the nucleic acidmodifying agent is a CRISPR-Cas system.11. The method of according to paragraph 9, wherein the CRISPR-Cassystem is a CRISPR-Cas9 system, a CRISPR-Cpf1 system, or a CRISPR-Cas13system.12. The method according to any of paragraphs 1 to 7, wherein the agentis administered in a combination treatment regimen comprising checkpointblockade therapy and/or adoptive cell therapy (ACT).13. The method according to paragraph 12, wherein the checkpointblockade therapy comprises anti-PD-1, anti-CTLA4, anti-PDL1, anti-TIM-3and/or anti-LAG3.14. The method according to any of paragraphs 1 to 13, wherein the agentis administered in a combination treatment regimen comprising aneoantigen vaccine.15. The method according to any of paragraphs 1 to 14, wherein thecancer expresses CLEC2D.16. The method according to any of paragraphs 1 to 15, wherein immunecells in the tumor microenvironment express KLRB1.17. The method of any of paragraphs 1 to 12, wherein the immune cellsare tumor infiltrating lymphocytes (TILs).18. The method according to any of paragraphs 1 to 13, wherein thecancer is glioblastoma multiforme (GBM), renal cancer, lungadenocarcinoma, or colon adenocarcinoma.19. An isolated T cell modified to comprise decreased expression oractivity of, or modified to comprise an agent capable of decreasingexpression or activity of KLRB1.20. The T cell according to paragraph 19, wherein the T cell is a CD8+ Tcell.21. The T cell according to paragraph 19, wherein the T cell is a CD4+ Tcell.22. The T cell according to any of paragraphs 19 to 21, wherein the Tcell is obtained from peripheral blood mononuclear cells (PBMCs).23. The T cell according to any of paragraphs 19 to 22, wherein the Tcell is an autologous T cell from a subject in need of treatment.24. The T cell according to any of paragraphs 19 to 23, wherein the Tcell is a TIL obtained from a subject in need of treatment.25. The T cell according to any of paragraphs 19 to 24, wherein the Tcell comprises a chimeric antigen receptor (CAR) or an exogenous T-cellreceptor (TCR).26. The T cell according to paragraph 25, wherein the exogenous TCR isclonally expanded in a tumor.27. The T cell according to paragraph 25 or 26, wherein the CAR or TCRis specific for a tumor antigen.28. The T cell according to paragraph 27, wherein the tumor antigen isEGFRvIII.29. The T cell according to paragraph 27, wherein the tumor antigen isselected from the group consisting of: B cell maturation antigen (BCMA);PSA (prostate-specific antigen); prostate-specific membrane antigen(PSMA); PSCA (Prostate stem cell antigen); Tyrosine-protein kinasetransmembrane receptor ROR1; fibroblast activation protein (FAP);Tumor-associated glycoprotein 72 (TAG72); Carcinoembryonic antigen(CEA); Epithelial cell adhesion molecule (EPCAM); Mesothelin; HumanEpidermal growth factor Receptor 2 (ERBB2 (Her2/neu)); Prostase;Prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M);Insulin-like growth factor 1 receptor (IGF-1R); gplOO; BCR-ABL(breakpoint cluster region-Abelson); tyrosinase; New York esophagealsquamous cell carcinoma 1 (NY-ESO—1); x-light chain, LAGE (L antigen);MAGE (melanoma antigen); Melanoma-associated antigen 1 (MAGE-A1); MAGEA3; MAGE A6; legumain; Human papillomavirus (HPV) E6; HPV E7; prostein;survivin; PCTA1 (Galectin 8); Melan-A/MART-1; Ras mutant; TRP-1(tyrosinase related protein 1, or gp75); Tyrosinase-related Protein 2(TRP2); TRP-2/INT2 (TRP-2/intron 2); RAGE (renal antigen); receptor foradvanced glycation end products 1 (RAGE1); Renal ubiquitous 1, 2 (RU1,RU2); intestinal carboxyl esterase (iCE); Heat shock protein 70-2(HSP70-2) mutant; thyroid stimulating hormone receptor (TSHR); CD123;CD171; CD19; CD20; CD22; CD26; CD30; CD33; CD44v7/8 (cluster ofdifferentiation 44, exons 7/8); CD53; CD92; CD100; CD148; CD150; CD200;CD261; CD262; CD362; CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319, and19A24); C-type lectin-like molecule-1 (CLL-1); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (Tn Ag);Fms-Like Tyrosine Kinase 3 (FLT3); CD38; CD138; CD44v6; B7H3 (CD276);KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2);Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen(PSCA); Protease Serine 21 (PRSS21); vascular endothelial growth factorreceptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growthfactor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4(SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16 (MUC16);epidermal growth factor receptor (EGFR); epidermal growth factorreceptor variant III (EGFRvIII); neural cell adhesion molecule (NCAM);carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit,Beta Type, 9 (LMP2); ephrin type-A receptor 2 (EphA2); Ephrin B2;Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; high molecularweight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside(OAcGD2); Folate receptor alpha; Folate receptor beta; tumor endothelialmarker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R);claudin 6 (CLDN6); G protein-coupled receptor class C group 5, member D(GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a;anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1(PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH);mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2);Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3(ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2(OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumorprotein (WT1); ETS translocation-variant gene 6, located on chromosome12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A(XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); CT(cancer/testis (antigen)); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53;p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin Bi; Cyclin Di; v-myc avian myelocytomatosisviral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog FamilyMember C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor(Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma AntigenRecognized By T Cells-1 or 3 (SART1, SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint-1, -2, -3 or -4 (SSX1, SSX2, SSX3, SSX4);CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1(LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300molecule-like family member f (CD300LF); C-type lectin domain family 12member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyteantigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); mousedouble minute 2 homolog (MDM2); livin; alphafetoprotein (AFP);transmembrane activator and CAML Interactor (TACI); B-cell activatingfactor receptor (BAFF-R); V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog (KRAS); immunoglobulin lambda-like polypeptide 1 (IGLL1); 707-AP(707 alanine proline); ART-4 (adenocarcinoma antigen recognized by T4cells); BAGE (B antigen; b-catenin/m, b-catenin/mutated); CAMEL(CTL-recognized antigen on melanoma); CAPi (carcinoembryonic antigenpeptide 1); CASP-8 (caspase-8); CDC27m (cell-division cycle 27 mutated);CDK4/m (cycline-dependent kinase 4 mutated); Cyp-B (cyclophilin B); DAM(differentiation antigen melanoma); EGP-2 (epithelial glycoprotein 2);EGP-40 (epithelial glycoprotein 40); Erbb2, 3, 4 (erythroblasticleukemia viral oncogene homolog-2, -3, 4); FBP (folate binding protein);, fAchR (Fetal acetylcholine receptor); G250 (glycoprotein 250); GAGE (Gantigen); GnT-V (N-acetylglucosaminyltransferase V); HAGE (helicoseantigen); ULA-A (human leukocyte antigen-A); HST2 (human signet ringtumor 2); KIAA0205; KDR (kinase insert domain receptor); LDLR/FUT (lowdensity lipid receptor/GDP L-fucose: b-D-galactosidase 2-a-Lfucosyltransferase); L1CAM (L1 cell adhesion molecule); MC1R(melanocortin 1 receptor); Myosin/m (myosin mutated); MUM-1, -2, -3(melanoma ubiquitous mutated 1, 2, 3); NA88-A (NA cDNA clone of patientM88); KG2D (Natural killer group 2, member D) ligands; oncofetal antigen(h5T4); p190 minor bcr-abl (protein of 190KD bcr-abl); Pml/RARa(promyelocytic leukaemia/retinoic acid receptor a); PRANE(preferentially expressed antigen of melanoma); SAGE (sarcoma antigen);TEL/AML1 (translocation Ets-family leukemia/acute myeloid leukemia 1);TPI/m (triosephosphate isomerase mutated); CD70; and any combinationthereof.30. The T cell according to any of paragraphs 19 to 29, wherein the Tcell is further modified to comprise decreased expression or activityof, or modified to comprise an agent capable of decreasing expression oractivity of a gene or polypeptide selected from the group consisting ofTOB1, RGS1, TARP, NKG7, CCL4 and any combination thereof.31. The T cell according to any of paragraphs 19 to 30, wherein the Tcell is activated.32. The T cell according to any of paragraphs 19 to 31, wherein the Tcell is modified using a CRISPR system comprising guide sequencesspecific to the target.33. The T cell according to paragraph 32, wherein the CRISPR systemcomprises Cas9, Cpf1 or Cas13.34. A population of T cells comprising T cells according to any ofparagraphs 19 to 33.35. A pharmaceutical composition comprising the population of T cellsaccording to paragraph 34.36. A method of treating cancer in a subject in need thereof comprisingadministering the pharmaceutical composition according to paragraph 35to the subject.37. The method of treatment according to paragraph 36, wherein thepopulation of cells are administered by infusion into the cerebralspinal fluid (CSF).38. The method of treatment according to paragraph 36, wherein thepopulation of cells are administered by injection into the cerebralspinal fluid (CSF) through the lateral ventricle.39. The method of treatment according to any of paragraphs 36 to 38,wherein the population of cells are administered in a combinationtreatment regimen comprising checkpoint blockade therapy.40. The method of treatment according to paragraph 39, wherein thecheckpoint blockade therapy comprises anti-PD-1, anti-CTLA4, anti-PDL1,anti-TIM-3 and/or anti-LAG3.41. The method according to any of paragraphs 36 to 40, wherein thecancer expresses CLEC2D.42. The method according to any of paragraphs 36 to 41, wherein tumorinfiltrating lymphocytes (TILs) in the cancer express KLRB1.43. The method according to any of paragraphs 36 to 42, wherein thecancer is glioblastoma multiforme (GBM).44. A method of generating a population of T cells for adoptive celltransfer, said method comprising:

-   -   a) obtaining a population of T cells;    -   b) delivering to the population of T cells a CRISPR system        comprising one or more guide sequences targeting KLRB1; and    -   c) activating the population of cells.        45. The method according to paragraph 44, wherein the CRISPR        system comprises Cas9, Cpf1 or Cas13.        46. The method according to paragraph 44 or 45, wherein the        CRISPR system is delivered as a ribonucleoprotein (RNP) complex        by electroporation.        47. The method according to any of paragraphs 44 to 46, wherein        activating comprises culturing the population of cells with αCD3        and αCD28 beads and IL-2.        48. The method according to any of paragraphs 44 to 47, further        comprising transducing the population of cells with a vector        encoding a chimeric antigen receptor (CAR) or an exogenous        T-cell receptor (TCR).        49. The method according to paragraph 48, wherein the vector        furthers encodes a detectable marker and the T cells expressing        a CAR or TCR are purified by sorting cells positive for the        detectable marker.        50. The method according to any of paragraphs 44 to 49, wherein        the T cells are obtained from PBMCs.        51. The method according to paragraph 50, wherein the PBMCs are        obtained from a subject in need of treatment.        52. The method according to any of paragraphs 44 to 49, wherein        the T cells are obtained from TILs obtained from a subject in        need of treatment.        53. A soluble KLRB1 protein or fragment thereof for use in the        treatment of cancer.        54. A KLRB1 antibody for use in the treatment of cancer.        55. A CLEC2D antibody for use in the treatment of cancer.        56. A method of assessing the efficacy of a therapeutic agent in        a subject afflicted with cancer, comprising

a) detecting the number of viable and/or proliferating cancer cells in asample from a subject;

b) administering a therapeutically effective amount of the agent to thesubject;

c) repeating step a) one or more times; and

d) comparing the number of viable and/or proliferating cancer cellsdetected in steps a) to those detected in step c),

wherein the therapeutic agent inhibits the interaction between KLRB1 andone or more natural binding partners of KLRB1, and

wherein the absence of, or a significant decrease in number of viableand/or proliferating cancer cells detected in step c) as compared to thenumber of viable and/or proliferating cancer cells detected in step a)indicates that the agent is effective.

57. The method of paragraph 56, wherein between steps a) and c) thesubject has undergone treatment, completed treatment for the cancer,and/or is in remission.58. The method of paragraph 56 or 57, wherein the samples in step a)and/or c) are ex vivo or in vivo samples.59. The method of any one of paragraphs 56-58, wherein the samples fromstep a) and/or c) are obtained from an animal model of the cancer.60. The method of any one of paragraphs 56-59, wherein the sample fromstep a) and/or c) is a portion of a single sample or pooled samplesobtained from the subject.61. The method of any one of paragraphs 56-60, wherein the samplecomprises cells, serum, peritumoral tissue, and/or intratumoral tissueobtained from the subject.62. The method of any one of paragraphs 56-61, further comprisingdetermining responsiveness to the agent by measuring at least onecriteria selected from the group consisting of clinical benefit rate,survival until mortality, pathological complete response,semi-quantitative measures of pathologic response, clinical completeremission, clinical partial remission, clinical stable disease,recurrence-free survival, metastasis free survival, disease freesurvival, circulating tumor cell decrease, circulating marker response,and RECIST criteria.63. The method of any one of paragraphs 56-62, wherein the cancer isglioblastoma multiforme (GBM), renal cancer, lung adenocarcinoma, orcolon cancer.64. A method for screening compounds which inhibit the interactionbetween KLRB1 and one or more natural binding partners of KLRB1comprising contacting (a) a cell expressing the one or more naturalbinding partners of KLRB1 with a KLRB1 protein; (b) a cell expressingKLRB1 with the one or more natural binding partners of KLRB1; (c) a cellexpressing the one or more natural binding partners of KLRB1 with a cellexpressing the one or more natural binding partners of KLRB1; or (d) aKLRB1 protein or the protein of one or more natural binding partners ofKLRB1, or biologically active portion thereof, with a test compound anddetermining the ability of the test compound to inhibit the interactionbetween KLRB1 and the one or more natural binding partners of KLRB1.65. A method for screening compounds comprising contacting (a) a cellexpressing one or more natural binding partners of KLRB1 with a KLRB1protein; (b) a cell expressing KLRB1 with the one or more naturalbinding partners of KLRB1; (c) a cell expressing the one or more naturalbinding partners of KLRB1 with a cell expressing the one or more naturalbinding partners of KLRB1; or (d) a KLRB1 protein or the protein of oneor more natural binding partners of KLRB1, or biologically activeportion thereof, with a test compound and determining the ability of thetest compound to inhibit one or more signaling activities selected fromthe group consisting of downregulating expression of cytototoxicproteins, downregulating expression of the activation marker CD69, anddownregulating cytokine production;

wherein the compounds inhibit the signaling activity resulting from theinteraction between KLRB1 and one or more natural binding partners ofKLRB1.

66. The method of paragraph 64 or 65, wherein the cell expressing KLRB1is a T cell selected from the group consisting of a CD4+ T cell, a CD8+T cell, and a CAR T cell.67. The method of any one of paragraphs 64-66, wherein the cellexpressing one or more natural binding partners of KLRB1 expressesCLEC2D, and wherein the cell is a cancer cell selected from the groupconsisting of a glioma cell, a renal cancer cell, a lung adenocarcinomacell, and a colon adenocarcinoma cell.68. A cell-based assay for screening compounds comprising contacting (a)a cell expressing the one or more natural binding partners of KLRB1 witha KLRB1 protein; (b) a cell expressing KLRB1 with the one or morenatural binding partners of KLRB1; or (c) a cell expressing the one ormore natural binding partners of KLRB1 with a cell expressing the one ormore natural binding partners of KLRB1, with a test compound anddetermining the ability of the test compound to inhibit the interactionbetween KLRB1 and the one or more natural binding partners of KLRB1,

wherein the compounds inhibit the interaction between KLRB1 and one ormore natural binding partners of KLRB1.

69. A cell-free assay for screening compounds comprising contacting oneor more natural binding partners of KLRB1 or KLRB1, or biologicallyactive portion thereof, with a test compound and determining the abilityof the test compound to inhibit the interaction between KLRB1 and theone or more natural binding partners, or biologically active portionthereof,

wherein the compounds inhibit the interaction between KLRB1 and one ormore natural binding partners of KLRB1.

70. A cell-based assay for screening compounds comprising contacting (a)a cell expressing one or more natural binding partners of KLRB1 with aKLRB1 protein; (b) a cell expressing KLRB1 with the one or more naturalbinding partners of KLRB1; or (c) a cell expressing the one or morenatural binding partners of KLRB1 with a cell expressing the one or morenatural binding partners of KLRB1, with a test compound and determiningthe ability of the test compound to inhibit one or more signalingactivities selected from the group consisting of downregulatingexpression of cytototoxic proteins, downregulating expression of theactivation marker CD69, and downregulating cytokine production,

wherein the compounds inhibit the signaling activity resulting from theinteraction KLRB1 and CLEC2D.

71. A cell-free assay for screening compounds comprising contacting oneor more natural binding partners of KLRB1 or KLRB1, or biologicallyactive portion thereof, with a test compound and determining the abilityof the test compound to inhibit one or more signaling activitiesselected from the group consisting of downregulating expression ofcytototoxic proteins, downregulating expression of the activation markerCD69, and downregulating cytokine production,

wherein the compounds inhibit the signaling activity resulting from theinteraction between KLRB1 and one or more natural binding partners ofKLRB1.

72. The assay of paragraph 70 or 71, wherein the cell expressing KLRB1is a T cell selected from the group consisting of a CD4+ T cell, a CD8+T cell, and a CAR T cell.73. The assay of any one paragraphs 70, 71, or 72, wherein the cellexpressing the one or more natural binding partners of KLRB1 is a cancercell selected from the group consisting of a glioma cell, a renal cancercell, a lung adenocarcinoma cell, and a colon adenocarcinoma cell.74. The method or assay of any one of paragraphs 1-73, wherein thecontacting or administering occurs in vivo, ex vivo, or in vitro.75. The method or assay of any one of paragraphs 1-74, wherein thecontacting or administering occurs in a subject and the subject is ananimal model of cancer, optionally wherein the animal model is a mousemodel.76. The method or assay of paragraph 75, wherein the subject is amammal.77. The method or assay of paragraph 76, wherein the mammal is a mouseor a human.78. The method or assay of any one of paragraphs 1-77, wherein the agentis administered in a pharmaceutically acceptable formulation.79. The method or assay of any one of paragraphs 1-78, wherein the oneor more natural binding partners of KLRB1 is CLEC2D.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1: KLRB1 is a Negative Immunoregulator inCancer-Infiltrating T Cells

Immune cells, such as cytotoxic T cells, interact with target cells,such as cancer cells, through cell surface receptor-ligand binding andcancers can evade immune cell activity by modulating the function ofsuch immune cells, such as by expressing a cell surface protein thatnegatively regulates immune function in the immune cell. It was desiredto identify new immunosuppressive mechanisms in GBM patients with aprimary focus on single-cell RNA-seq (scRNA-seq) analysis of T cellsdirectly isolated from surgically resected lesions (FIGS. 1A-1B). Inparticular, T cell infiltrates of GBM tumors have low T cell activationdespite only moderate expression of PD-1 (FIGS. 2A-2B), indicating thatadditional immunosuppressive mechanisms are active in GBM tumors.

As described in detail below, based on scRNA-seq of GBMtumor-infiltrating T cells, it was determined herein that one of the topgene products was KLRB1 (also known as CD161), a C-type lectin proteinthat binds to CLEC2D (FIG. 3). KLRB1 was also determined to be highlyexpressed in clonally expanded CD8+ and CD4+ T cell clones (FIGS. 4B, 5,6, 21A, and 21B). KLRB1 binds CLEC2D with low binding affinity, but withfast kinetics. Binding of CLEC2D to the KLRB1 receptor inhibits thecytotoxic function of NK cells as well as cytokine secretion (Rosen etal. (2005) J. Immunol. 175:7796-7799). KLRB1 is only expressed by smallsubpopulations of human blood T cells, and consequently little is knownabout the function of this receptor in T cells (Kirkham and Carlyle(2014) Front. Immunol. 5:214). However, it was demonstrated herein thatKLRB1 expression was induced in T cells within the GBM microenvironment.An immunohistochemistry study demonstrated that CLEC2D (also calledLLT1) was expressed by human gliomas, with expression increasing withWHO grade of malignancy. In contrast, there was little labeling with aCLEC2D antibody in sections from normal human brain tissue (Roth et al.(2007) Cancer Res. 67:3540-3544). These conclusions are supported by ananalysis of TCGA RNA-seq data which demonstrated significantly increasedexpression of CLEC2D in GBM compared to normal brain tissue(p=5.1×10⁻¹¹). This analysis also highlighted increased expression ofCLEC2D relative to the corresponding normal tissue in many other cancertypes, including all types of renal cancer (p<2×10⁻¹⁶ for KIRC), lungadenocarcinoma (p=5.5×10⁻¹¹), colon adenocarcinoma (p=3.1×10⁻¹²) andother cancers. These data indicate that KLRB1 functions as an inhibitoryreceptor for human T cells by binding to the CLEC2D ligand on tumorcells. This is supported by data from a humanized mouse model of GBMwhich demonstrated that inactivation of the KLRB1 gene in primary humanT cells greatly enhanced their cytotoxic function within tumors.

Very little is known about the functional state of T cells in GBM. TheCNS is believed to create a unique microenvironment that impacts T cellfunction by distinct mechanisms. In order to address this challenge,scRNA-seq was performed in 5 untreated IDH-wildtype GBM samples, and-2,000 T cells (that passed QC metrics) were profiled, spanning theexpected main classes of T cells (CD8, CD4, Tregs, naïve, and cytotoxicT cells,) (FIG. 4A). This approach not only provided insights into thefunctional state of these cells, but also enables identification of theclonally expanded T cells through reconstruction of the alpha and betachain of T cell receptors (TCRs) (Stubbington et al. (2016) Nat. Methods13:329-332). In GBM, both dysfunctional T cells that express multipleinhibitory receptors (TIGIT, PD1, TIM3, CTLA4) but also T cells that arefunctional based on expression of multiple genes required for T cellcytotoxicity (GZMB, PRF1, NKG7, etc.) were observed. Analysis ofclonally expanded T cells was emphasized because such T cells haveundergone proliferation due to (tumor) antigen recognition. Thisanalysis highlighted KLRB1 (also known as CD161) as being highlyexpressed in clonally expanded T cells (FIG. 4B). In contrast, both PD-1(encoded by PDCD1) and CTLA-4 ranked lower in this analysis. KLRB1 isonly expressed by small subpopulations of human blood T cells, andconsequently little is known about the function of this receptor in Tcells. The scRNA-seq data indicate that KLRB1 expression is induced inclonal T cells (both CD4 and CD8 T cells) within the GBMmicroenvironment (FIGS. 4B, 5, 6, 21A, and 21B).

Example 2: The KLRB1 Pathway, Including the KLRB1—CLEC2D Interaction, isan Immunotherapy Target for Cancers, Including GBM

Given that KLRB1 was identified as a candidate target in human GBM, itsfunctional significance in human T cells rather than in a moreconvenient murine model system was validated. Human GBM cells wereimplanted orthotopically into the CNS of immunodeficient NSG mice bystereotactic injection. Two human GBM cell lines (U87 and D-270 MG) weretested for CLEC2D expression and it was found that CLEC2D was highlyexpressed by both tumor cell lines (FIGS. 7A and 7B). T cell-mediatedimmunity against such tumors requires activation of T cells througheither the T cell receptor (TCR) or an introduced chimeric antigenreceptor (CAR). Applicants expressed the NY-ESO—1 antigen in U87 GBMcells and introduced the NY-ESO—1 TCR in human peripheral blood T cells.Genetic editing of these T cells was performed by electroporation ofCas9/gRNA complexes (FIG. 8A) (Schumann et al. (2015) Proc. Natl. Acad.Sci. USA 112:10437-10442). This technique was carefully optimized inorder to routinely provide editing efficiencies of >90% with a suitablegRNA (FIG. 8B). This approach thus created T cells specific for NY-ESO—1that can be edited with KLRB1 or control gRNAs. In vitro studiesdemonstrated that T cells edited with the KLRB1 gRNA kill pre-culturedtumor cells (FIG. 9). In addition, the edited T cells secretedsignificantly higher levels of IL-2 when co-cultured with the U87 GBMcell line (FIG. 10B), as well as IFN-gamma (FIG. 10A), supporting theconclusion that the KLRB1 receptor delivers an inhibitory signal in Tcells.

A recent clinical trial demonstrated that CAR T cells induced a completeresponse in one patient with multi-focal GBM (sustained for 7.5 months)when CAR T cells were infused into the CSF (Brown et al. (2016) N. Engl.J. Med. 375:2561-2569). Based on this report, the edited NY-ESO—1 TCRtransduced T cells were delivered into the CSF of mice by stereotacticinjection into the lateral ventricle (FIG. 11). Control injections witha dye confirmed the effectiveness of this technique (FIG. 12). Theimpact of injected NY-ESO—1 TCR T cells was tracked by bioluminescence,and reduced tumor growth or tumor regression in mice injected wasobserved with KLRB1-edited T cells, but not with control-edited T cells(LacZ gRNA) (FIGS. 13 and 14A-16C). Moreover, mice injected withKLRB1-edited T cells demonstrated improved survival (FIG. 19).

On day 8 following T cell transfer, single cell suspensions weregenerated from isolated tumors for in-depth characterization oftumor-infiltrating T cells. The activation state of CD8+ T cells thatexpressed the NY-ESO—1 TCR was greatly enhanced by KLRB1 editing (FIGS.15-16). These cells expressed higher levels of the cytotoxicityproteins, granzyme B and perforin, the CD69 activation marker, and keycytokines (FIG. 17). Similar results were obtained with CD4+ T cells(FIG. 18). Interestingly, KLRB1-edited CD4+ T cells expressed granzymeB, indicating that inactivation of this inhibitory receptor can enabletheir differentiation into cytotoxic effector cells.

Example 3: The Importance of the KLRB1 Pathway, Including theKLRB1—CLEC2D Interaction, in Other Major Classes of Human Gliomas andAcross Human Cancers

In order to further confirm whether KLRB1 and CLEC2D are expressed inmost cases of IDH-wildtype GBM or whether there are additional andalternate regulatory programs for T cell function, the studies describedabove were extended to additional cases of untreated IDH-wildtype GBM byscRNA-seq, focusing primarily on the immune compartment. Data weregenerated with two complementary platforms. For the first platform,massively parallel droplet based scRNA-seq (10×Genomics platform) wasused, which enables analysis of several thousand cells per sample.Immune cells (CD45+) were separated from non-immune and tumor cells(CD45−) by magnetic beads and single cell libraries were generated fromeach population. For the second platform, flow cytometry was also usedto sort T cells (e.g., CD45+, CD3+, CD4+ or CD8+) into 96-well or384-well plates for full-length whole transcriptome amplification. Therobust modified SMART-Seq2 method can be used as an effective andreproducible protocol to obtain full-length transcript information,which is key to map scRNA-Seq reads to TCR transcripts (Picelli et al.(2014) Nat. Protoc. 9:171-181). The relationship between T cell statesand clonal expansion was analyzed. To reduce costs, 384 single-celllibraries were pooled in one NextSeq500 run, providing a depth ofsequencing of ˜1.5 mio reads/cell, an optimized range based onsaturation experiments.

In order to confirm whether the KLRB1 pathway, including theKLRB1—CLEC2D interaction, is relevant in other classes of human gliomas,the methods described above were applied to additional human gliomatypes. In addition to IDH-wildtype adult GBM, extensive single celldatasets in pediatric glioblastoma, IDH-mutant oligodendroglioma andastrocytoma as well as histone H3.3 mutant midline pediatric gliomashave been generated (Tirosh et al. (2016) Science 352:189-196;Venteicher et al. (2017) Science 355:eaai8478). The existing pipelineswere leveraged to continue to collect and characterize all major classesof adult and pediatric gliomas, focusing on the T cells compartment. Anadditional 10 cases of diffuse gliomas across major classes wereaccrued. This analysis is expected to shed additional light on themechanisms that govern T cell function in primary parenchymal braintumors. IDH-mutant gliomas (oligodendroglioma and astrocytoma) are ofparticular interest to compare to IDH-wildtype GBM given the immunemodulatory effect of the oncometabolite 2-hydroxy-glutarate (2-HG) thataccumulates in the microenvironment of IDH-mutant gliomas (Kohanbash etal. (2017) J. Clin. Invest. 127:1425-1437).

In order to confirm whether the KLRB1 pathway, including theKLRB1—CLEC2D interaction, is relevant in non-CNS malignancies, acollection of human cancers being profiled and available at thesingle-cell level was used. These include, without limitation, melanoma,colon carcinoma, breast cancer, head-and-neck cancer, synovial sarcoma,and lung adenocarcinoma. These single-cell datasets extend the bulkanalyses that were performed in TCGA cohorts. For each of thesemalignancies, the single-cell profiles of both malignant cell andnonmalignant cells in the microenvironment were interrogated andquestions such as in which of these tumors KLRB1 and CLEC2D areexpressed, in which cells in the tumor microenvironment KLRB1 and CLEC2Dare expressed, whether expression of CLEC2D and/or KLRB1 associated withmarkers of T cell dysfunction, and whether regulatory pathways thatcontrol expression of both receptor and ligand, can be inferred wereaddressed.

Example 4: Impact of KLRB1 and CLEC2D on T Cell Mediated Tumor Immunity

To further confirm that KLRB1 acts as an inhibitory receptor fortumor-infiltrating T cells additional experiments may be performed. Forexample, additional survival experiments may be performed using two GBMcell lines: the U87 cell line described above and the D-270 MG gliomacell line which grow in a highly infiltrative manner in the CNS, a majorfeature of GBM (Miao et al. (2014) PLoS One 9:e94281). T cell dose canbe optimized and tumor growth tracked by bioluminescence. The functionalstate of KLRB1 versus control edited T cells may be assessed atdifferent time points by flow cytometry, with an emphasis on cytotoxicfunction (perforin, granzyme B) and expression of other inhibitoryreceptors (PD-1, TIM-3 and LAG3). The D-270 MG model also provides anopportunity to examine if KLRB1 edited T cells are more effective intargeting highly infiltrative tumor cells (multi-colorimmunofluorescence analysis of tissue sections).

In order to confirm that CLEC2D expressed by glioma cells inhibits thefunction of tumor-infiltrating T cells, the CLEC2D gene may beinactivated in both U87 and D-270 MG glioma cell lines. Editing may thenbe performed by electroporation of Cas9/gRNA complexes (CLEC2D orcontrol gRNAs), and loss of CLEC2D surface expression confirmed by flowcytometry. Comparison of tumor-infiltrating T cells fromCLEC2D-deficient or control-edited tumors demonstrates may be used todemonstrate that loss of CLEC2D expression enhances T cell accumulationwithin tumors and their cytotoxic function (expression of perforin andgranzyme B). Whether CLEC2D-deficient tumors are more readily rejectedby transferred T cells may also be examined.

Whether editing of KLRB1 or CLEC2D result in similar changes in T cellfunction is useful because additional ligands for KLRB1 or receptors forCLEC2D may exist. For example, there are two ligands (PD-L1 and PD-L2)for the PD-1 receptor (Baumeister et al. (2016) Annu. Rev. Immunol.34:539-573). scRNA-seq was performed on T cells isolated fromorthotopically implanted tumors, and whether inactivation of CLEC2D intumor cells or KLRB1 in T cells induces similar changes in the T celltranscriptome may be examined.

Antibody-based targeting of KLRB1 or CLEC2D to confirm enhancement ofanti-tumor function of T cells may be performed using therapyexperiments with anti-KLRB1 antibodies and/or anti-CLEC2D antibodies,such as the fully human KLRB1- or CLEC2D-specific antibodies describedin Example 6 below. NSG mice lack B cells, and mouse anti-humanantibodies are therefore not an issue. These antibodies (200 μg/mouse,twice per week) were injected when tumors were established (−day 7) andmice may be randomized to treatment and isotype control antibody groupsbased on the bioluminescence signal. The blood brain barrier is leaky inGBM tumors, and antibodies can therefore diffuse into the tumormicroenvironment (Ait-Belkacem et al. (2014) MAbs 6:1385-1393).

Clinical trials have thus far failed to demonstrate substantial clinicalbenefit with PD-1 blocking antibodies in patients with GBM, indicatingthat other inhibitory signals may limit the effectiveness ofimmunotherapy. The humanized model described above provides anopportunity to confirm whether targeting of KLRB1 synergizes withblockade of PD-1 or other inhibitory receptors (such as TIM-3 or LAG3).This may be addressed by transfer of KLRB1-edited T cells or injectionof a fully human KLRB1 blocking antibody. scRNA-seq may be alsoperformed on tumor-infiltrating T cells to define the transcriptionalprograms that are impacted by these combinatorial strategies.

Only a small subset of peripheral blood T cells express KLRB1,indicating that its expression is induced in the tumor microenvironment.To identify signals from tumor microenvironment that induce KLRB1expression in T cells, the following candidate molecules may beinvestigated: a) Immunosuppressive cytokines including IL-10 and TGFβ,b) Inflammatory cytokines that promote tumor growth including TNFα, IL-6and IL-1, c) Immunosuppressive small molecule mediators, includingadenosine (degradation product of ATP in hypoxic tumors), tryptophancatabolites produced by IDO/TDO and prostaglandin E2. This analysis canprovide alternative strategies for interfering with this pathway usingavailable drugs (such as antibodies targeting TNFα, IL-6, or IL-1; smallmolecules targeting the adenosine receptor or IDO/TDO; etc.).

Example 5: Generation of Fully Human Therapeutic Antibodies Specific forKLRB1 and CLEC2D

Fully human antibodies against both CLEC2D and KLRB1 will be isolatedand affinity matured by interrogating a 109-member scFv fragment librarydisplayed on the surface of yeast. An antibody library with unusuallystrong “developability” characteristics has been constructed (FIGS.20A-20C) (Jain et al. (2017) Proc. Natl. Acad. Sci. USA 114:944-949).

CLEC2D and KLRB1 will be expressed as Fc fusion proteins and purified byprotein A affinity chromatography. Both proteins form homodimers andfusion of the Fc region is therefore expected to enhance proteinexpression and stability. A library will be screened for lead bindersagainst human CLEC2D and KLRB1, using magnetic bead screening and flowcytometry as described previously in Chao et al. (2006)Nat. Protoc.1:755-768. Once an initial set of binders is found, the subset thatcompetes with CLEC2D/KLRB1 binding will be identified by adding anexcess of the unlabeled binding partner (e.g., for binders tobiotinylated KLRB1, excess unlabeled CLEC2D was added and those cloneswith decreased binding were selected). As a further criterion, clonesthat were cross-reactive to murine CLEC2D and KLRB1 will be selected, toenable therapeutic studies with murine syngeneic and GEMM tumors. Ifcross-reactive clones are not identified in the first round, thisspecificity may be introduced by directed evolution, using mutagenesisand yeast display. The initial affinity of scFvs isolated from thisrepertoire requires improvement to reach the sub-nanomolar affinitygenerally expected for antibody drug leads. Such affinity maturation isstraightforward and robust in the yeast display system (Boder et al.(2000) Proc. Natl. Acad. Sci. USA 97:10701-10705; Graff et al. (2004)Prot. Eng. Des. Sel. 17:293-304).

The scFvs exhibiting desirable affinities and specificities will bereformatted for expression as human IgG4 antibodies, a non-activatingisotype most often used for drugs with purely antagonistic mechanisms ofaction (Beers et al. (2016) Blood 127:1097-1101). Although this isexpected to be the preferred approach, human IgG1 versions will also beconstructed to determine whether an unexpected cell clearance mechanismalso has therapeutic value, as was found for the anti-CTLA4 antibodyipilimumab, which was intended to block CTLA4/B7 interactions butdepends upon Treg clearance in murine systems (Selby et al. (2013)Cancer Immunol. Res. 1:32-42). As was determined in the examplesdescribed above, those cells expressing these targets will indicatewhether an activating or inactive isotype is preferable. By analogy toanti-PD-L1 antibodies, where the target is expressed on both tumor andmyeloid cells, an activating isotype is believed to contribute innateeffector functions targeting tumor cells expressing CLEC2D.

Example 6: GBM-Infiltrating T Cells Express High Levels of CD161

A surgically-resected recurrent glioblastoma was obtained on ice within30 minutes of lesion excision from the patient. The tumor specimen wasmechanically disrupted into small pieces with a disposable, sterilescalpel and further dissociated into a single cell suspension using theenzymatic brain dissociation kit (P) from Miltenyi Biotec (BergischGladbach, Germany), following the manufacturer's protocol. Fc receptorblocking was performed on the total cell suspension using Human TruStainFcX (Biolegend, San Diego, Calif.). The cell suspension was subsequentlystained for flow cytometry using antibodies against CD45-BV605,CD3-BV510, CD4-PE/Cy7, CD8-PerCP/Cy5.5, Exclusion panel-APC (CD14, CD64,CD163, CD15 and CD66b). The tumor cell suspension was next spiked with0.5 μM Calcein AM to enable gating of live cells. Results are shown inFIGS. 25A and 25B.

Results in FIGS. 33A and 33B illustrate that KLRB1 is expressed at ahigher level by T cells in GBM compared to melanoma. In FIG. 33A, geneexpression was averaged across all T cells. The top 10 genes upregulatedin GBM versus melanoma (data correlated with FIG. 33A) are shown inTable 3. In FIG. 33B, gene expression was averaged across CD8_CD8NK Tcells. The top 10 genes upregulated in GBM versus melanoma (datacorrelated with FIG. 33B) are shown in Table 4.

TABLE 3 Top 10 genes upregulated in GBM vs. melanoma identified using ttest. p_val avg_logFC pct. 1 pct. 2 KLRB1 9.09E−101 1.38724334 0.3480.175 CDKN1A 4.26E−148 1.33409656 0.303 0.099 CD55 2.71E−116 1.329579450.44 0.307 DPP4 1.11E−88  1.32639616 0.217 0.082 TNFAIP3 2.6757607241.28885779 0.892 0.723 PDE4B 4.76E−248 1.28865101 0.636 0.351 IFNGR18.96E−72  1.28262488 0.3 0.16 SC5DL 2.12E−76  1.21898706 0.624 0.74 CREM5.39E−259 1.21312708 0.668 0.408 ANXA1 1.84E−120 1.15712903 0.577 0.381

TABLE 4 Top 10 genes upregulated in GBM vs. melanoma identified using ttest. p_val avg_logFC pct. 1 pct. 2 KLRB1 1.93E−31 1.6305855 0.255 0.094TNFAIP3  1.72E−154 1.46263717 0.91 0.714 IFNGR1 6.40E−31 1.39597237 0.330.138 CD55 2.64E−44 1.37445102 0.465 0.292 PDE4B 1.34E−82 1.341312440.609 0.294 CXCR4  1.06E−199 1.29841242 0.98 0.802 ANXA1 9.00E−611.26421189 0.619 0.363 YPEL5 2.28E−65 1.25902478 0.68 0.428 CCNH8.03E−39 1.2266631 0.513 0.313 GPR183 9.07E−30 1.19547626 0.334 0.156

Example 7: Isolation of a CD161+ T Cell Population from Primary Human TCells for Functional Studies

FIG. 35A illustrates the method used to obtain a CD161+ T cellpopulation suitable for functional studies. Primary human T cells wereisolated by negative selection from peripheral blood mononuclear cells(PBMC) and stained for fluorescence activated cell sorting (FACS) withpreconjugated antibodies that recognize CD3-APC, Vα7.2-BV780, andCD161-Percp/Cy5.5. CD161+Vα7.2− T cells were sorted and cultured inhuman recombinant IL-2 (30U/ml) for functional studies. Results areshown in FIGS. 35B and 35C.

FIG. 36 illustrates the strategy used for high efficiency gene knockoutin human T cells. Primary CD161+Vα7.2− human T cells were electroporatedwith two ribonucleoprotein complexes (Cas9 [20μM]+gRNA [60μM]) directedagainst KLRB1 and the T cell receptor alpha constant chain locus (TRAC).T cells were immediately cultured with human dynabeads and IL-2(30U/ml). A control population of CD161+Vα7.2− human T cells wereelectroporated with gRNAs directed against LacZ and TRAC.

High efficiency gene knockout in human T cells was detected as follows.Genomic DNA (gDNA) was extracted from CD161+Vα7.2− human T cells 3 daysafter electroporation with ribonucleoproteins (RNP) directed againstKLRB1 and TRAC. The gDNA region of the anticipated RNP cutsite wasamplified by PCR that included 400 nucleotide flanking regions on eitherside of the cutsite. Sanger sequencing was performed on gel purified PCRproduct and analyzed by Tracking of Indels by Decomposition (TIDE)software to determine the efficiency of KLRB1 or TRAC editing comparedto LacZ control edited gDNA. Results of this experiment are shown inFIGS. 37A and 37B.

Example 8: Sorted Primary T Cells Retain Phenotype Long Term

Primary TRAC and KLRB1 (or LacZ control) edited CD161+Vα(7.2− human Tcells were transduced with a lentiviral construct (MOI 15) that resultsin the expression of the NY-ESO—1 [clone 1G4] T cell receptor (NYE_TCR)that expresses an HA-tag on the extracellular domain of the alpha chainand a PC-tag on the beta chain (FIG. 38A). Three days following thelentiviral infection, NYE_TCR+ T cells (HA-tag+) were sorted andexpanded in human IL-2 and human dynabeads. FIGS. 38B and 38B show thatT cell populations retain their sorted phenotype over a long-term invitro culture for 21 days. Control edited T cells (FIG. 38B) or KLRB1edited T cells (FIG. 38C) express high levels of NY-ESO—1 TCR.

Example 9: KLRB1 Deficient T Cells Exhibit Increased FunctionalPhenotype

The endogenous T-cell receptor (TRAC) and KLRB1 or LacZ (control) wereknocked out in primary human T cells by the electroporation ofgene-targeting RNPs (as described). The edited T cells were nexttransduced with lentivirus to express the NY-ESO—1 high affinity TCR(NYE_TCR). The engineered T cells were then cocultured with U87MG cellsthat express NY-ESO—1 peptide (U87-NYEP) at 1:2 effector to target ratiofor 24 hours or 48 hours. Cells were stained with preconjugatedantibodies that recognize ZombieUV (viability), CD3-BV510, CD8-BV650,CD4-APC/Cy7, GranzymeB-APC, Perforin-PE, CD69-BV421, IFNγ-BV711,TNFα-PE/Cy7, and IL-2-FITC and analyzed by flow cytometry. Results for24 hour co-cultures are shown in FIGS. 39A and 39B and results for 48hour co-cultures are shown in FIGS. 40A and 40B.

Example 10: Inactivation of the KLRB1 Gene Enhances Cytokine Productionby T Cells

The endogenous T-cell receptor and KLRB1 or LacZ (control) were knockedout in primary human T cells by the electroporation of gene-targetingRNPs (as described) were next transduced with lentivirus to express theNYE_TCR. The engineered NYE_TCR T cells were next cocultured withU87-NYEP tumor cells at the indicated effector to target ratios. Thesupernatants were removed from the cocultures at 72h and analyzed byELISA for IFNγ and IL-2. Similar data were observed at 24h and 48h.Results are shown in FIGS. 41A and 41B.

Example 11: Inactivation of the KLRB1 Gene Greatly Reduces PD-1Expression by T Cells

Engineered NYE-ESO—1 TCR+ T cells (NYE_TCR) that were edited for LacZ orKLRB1, were examined by flow cytometry at 48h or 72h following coculturewith U87MG cells that express NY-ESO—1 peptide at the indicated effectorto target ratios. Cells were stained with ZombieUV (viability),CD3-BV510, CD8-BV650, CD4-APC/Cy7, and PD-1-PE/Dazzle594. KLRB1 editedCD8+ T cells expressed significantly less PD-1 than control (LacZ)edited T cells. Results are shown in FIGS. 42A and 42B.

Example 12: Humanized Mouse Model to Study Effect of KLRB1 Edited TCells Against Human GBM Cells

A schematic of the stereotactic surgical procedures for xenograft modelof GBM is shown in FIG. 43. Mice were secured into a stereotactic deviceand 1.5×10{circumflex over ( )}4 U87MG cells that were transduced toexpress the NY-ESO—1 peptide (U87-NYEP) were injected into the leftstriatum. A second stereotactic surgery was performed 7 days later and0.4×10{circumflex over ( )}6 NYE_TCR+ T cells were injected into theright lateral ventricle.

Implanted tumors were removed from NSG mice 6 days after contralateralcerebroventricular injection of NYE_TCR⁺ T cells for analysis by flowcytometry. Individual tumors were dissociated into single cellsuspensions and incubated with protein transport inhibitors for 4 hours.All cells were stained with antibodies for flow cytometry: ZombieUV(viability), CD3-BV510, CD8-BV650, CD4-APC/Cy7, CD161-PerCP/Cy5.5,CD69-BV421, and PD1-PE/Dazzle594. Flow cytometry analysis was performedusing FlowJo version 10.5.3. As shown in FIG. 44, KLRB1 edited human Tcells exhibit increased activation and decreased inhibitory markers invivo, such as decreased expression of PD-1.

Seven days following the implantation of 1.5×10{circumflex over ( )}4U87-NYEP⁺ cells, 0.4×10{circumflex over ( )}6 NYE_TCR⁺ T cells that wereedited for TRAC and KLRB1 (or LacZ control) were administered into theright lateral ventricle by stereotactic injection. Animals were observedfor survival over 45 days. As shown in FIG. 45, KLRB1 edited T cellssignificantly improve mouse survival in xenograft model of GBM.

Various modifications and variations of the described methods,pharmaceutical compositions, and kits of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific embodiments, it will be understood that it iscapable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention. This application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure come within known customarypractice within the art to which the invention pertains and may beapplied to the essential features herein before set forth.

1. A method of treating a disease characterized by increased expressionof killer cell lectin like receptor (KLRB1) in immune cells, comprisingadministering to a subject in need thereof one or more therapeuticagents in an amount sufficient to either: (i) block binding of theprotein encoded by KLRB1 (CD161) to one or more CD161 ligands; (ii)reduce expression of KLRB1; (iii) reduce expression of one or more genesencoding one or more CD161 ligands (iv) block binding of CLEC2D to areceptor of CLEC2D other than KLRB1, or any combination thereof.
 2. Themethod of claim 1, wherein the one or more agents comprises an antibody,or fragment thereof, that binds CD161, preferably, wherein the antibodyis a humanized or chimeric antibody: or wherein the one or more agentscomprise an antibody, or fragment thereof, that binds to the one or moreCD161 ligands, preferably, wherein the antibody is a humanized orchimeric antibody: or administering a soluble CD161 protein, or fragmentthereof, that binds to one or more of the CD161 ligands.
 3. (canceled)4. (canceled)
 5. The method of claim 1, wherein reducing expression ofKLRB1, or expression of one or more genes encoding one or more CD161ligands, comprises administering a programmable nucleic acid modifyingagent configured to reduce expression of KLRB1, or reduce expression ofone or more or more genes encoding one or more CD161 ligands,preferably, wherein the programmable nucleic acid modifying agent is aCRISPR-Cas, a zinc finger, a TALE, or a meganuclease, more preferably,wherein the CRISPR-Cas is a CRISPR-Cas9, a CRISPR-Cas12, a CRISPR-Cas13,or a CRISPR-Cas14.
 6. (canceled)
 7. (canceled)
 8. The method of claim 1,wherein the disease is cancer, preferably, wherein the cancer ischaracterized by increased expression of a KLRB1 ligand by cancer cellsor other cells in the tumor microenvironment; or wherein one or moreimmune cell types in the tumor microenvironment are characterized byincreased expression of KLRB1, more preferably, wherein the one or moreCD161 ligands comprises CLEC2D; or wherein the one or more immune cellsare tumor infiltrating lymphocytes (TILs), including CD4 T cells, CD8 Tcells and NK cells: or wherein the cancer is a carcinoma, sarcoma,leukemia, lymphoma, myeloma, brain cancer, or spinal cord cancer. 9.(canceled)
 10. (canceled)
 11. (canceled)
 12. (canceled)
 13. The methodof claim 8, wherein the one or more agents are administered in acombination treatment regimen comprising checkpoint blockade therapy,vaccines, targeted therapies, radiation therapy, chemotherapy, and/oradoptive cell therapy (ACT), preferably, wherein the checkpoint blockadetherapy comprise anti-PD-1, anti-CTLA4, anti-TIM-3 and/or anti-LAG3; orwherein the vaccine is a neoantigen vaccine or other cancer vaccine. 14.(canceled)
 15. (canceled)
 16. The method of claim 1, wherein the diseaseis an infectious disease, preferably, wherein the infectious disease isa chronic infection disease, more preferably, wherein the infectiousdisease is a chronic viral infection, a chronic bacterial infection, ora chronic parasitic infection, more preferably, wherein the chronicviral infection is HIV, hepatitis B, hepatitis C: or wherein the chronicbacterial infection is tuberculosis, lyme disease, meningitis, Q fever,ehrlichiosis, bacterial vaginosis, pelvic inflammatory disease,rheumatic fever; or wherein the chronic parasitic infection is malaria,Chagas disease, or isosporiasis.
 17. (canceled)
 18. (canceled) 19.(canceled)
 20. (canceled)
 21. (canceled)
 22. The method of claim 16,wherein the bacteria infection is a severe bacterial infection of theintestine, and wherein the one or more agents are administered in anamount sufficient to enhance MAIT cell function.
 23. The method of claim16, wherein the disease is a latent HIV infection, preferably, whereinthe one or more therapeutic agents are administered in combination withone or more HIV therapeutic agents, such as one or more anti-retroviralagents.
 24. (canceled)
 25. A method of treating a chronic inflammatorydiseases comprising administering to a subject in need thereof one ormore agents in an amount sufficient to either increase expression ofKLRB1 and/or increase expression of one or more genes encoding one ormore CD161 ligands, or to activate or stimulate cell signaling throughKLRB1, preferably, wherein the one or more agents is a agonisticantibody of CD161: or wherein the one or more CD161 ligands comprisesCLEC2D; or wherein the chronic inflammatory diseases comprises anautoimmune disease.
 26. (canceled)
 27. (canceled)
 28. (canceled)
 29. Anisolated T cell modified to comprise decreased expression or activityof, or modified to comprise an agent capable of decreasing or increasingexpression of KLRB1 or activity of CD161, preferably, wherein the T cellis a CD8+ T cell or CD4+ T cell.
 30. (canceled)
 31. (canceled)
 32. The Tcell according to claim 29, wherein the T cell is obtained fromperipheral blood mononuclear cells (PBMCs); and/or wherein the T cell isan autologous T cell from a subject in need of treatment; and/or whereinthe T cell is a TIL obtained from a subject in need of treatment; and/orwherein the T cell comprises a chimeric antigen receptor (CAR) or anexogenous T-cell receptor (TCR), preferably, wherein the exogenous TCRis clonally expanded in a tumor, and/or wherein the CAR or TCR isspecific for a tumor antigen.
 33. (canceled)
 34. (canceled) 35.(canceled)
 36. (canceled)
 37. (canceled)
 38. The T cell according toclaim 32, wherein the tumor antigen is EGFRvIII, Her2, or other tumorsurface antigen; or wherein the tumor antigen is selected from the groupconsisting of; B cell maturation antigen (BCMA); PSA (prostate-specificantigen); prostate-specific membrane antigen (PSMA); PSCA (Prostate stemcell antigen); Tyrosine-protein kinase transmembrane receptor ROR1;fibroblast activation protein (FAP); Tumor-associated glycoprotein 72(TAG72); Carcinoembryonic antigen (CEA); Epithelial cell adhesionmolecule (EPCAM); Mesothelin; Human Epidermal growth factor Receptor 2(ERBB2 (Her2/neu)); Prostase; Prostatic acid phosphatase (PAP);elongation factor 2 mutant (ELF2M); Insulin-like growth factor 1receptor (IGF-1R); gplOO; BCR-ABL (breakpoint cluster region-Abelson);tyrosinase; New York esophageal squamous cell carcinoma 1 (NY-ESO-1);κ-light chain, LAGE (L antigen); MAGE (melanoma antigen);Melanoma-associated antigen 1 (MAGE-A1); MAGE A3; MAGE A6; legumain;Human papillomavirus (IPV) E6; HIPV E7; prostein; survivin; PCTA1(Galectin 8); Melan-A/MART-1; Ras mutant; TRP-1 (tyrosinase relatedprotein 1, or gp75); Tyrosinase-related Protein 2 (TRP2); TRP-2/INT2(TRP-2/intron 2); RAGE (renal antigen); receptor for advanced glycationend products 1 (RAGE1); Renal ubiquitous 1, 2 (RU1, RU2); intestinalcarboxyl esterase (iCE); Heat shock protein 70-2 (HSP70-2) mutant;thyroid stimulating hormone receptor (TSHR); CD123; CD171; CD19; CD20;CD22; CD26; CD30; CD33; CD44v7/8 (cluster of differentiation 44, exons7/8); CD53, CD92, CD100; CD148; CD150; CD200; CD261; CD262; CD362; CS-1(CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-likemolecule-1 (CLL-1); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (Tn Ag);Fms-Like Tyrosine Kinase 3 (FLT3); CD38; CD138; CD44v6; B7H3 (CD276);KIT (CD 117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2);Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen(PSCA); Protease Serine 21 (PRSS21); vascular endothelial growth factorreceptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growthfactor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4(SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16 (MUC16);epidermal growth factor receptor (EGFR); epidermal growth factorreceptor variant III (EGFRvIII); neural cell adhesion molecule (NCAM);carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit,Beta Type, 9 (LMP2); ephrin type-A receptor 2 (EphA2); Ephrin B2;Fucosyl GMI; sialyl Lewis adhesion molecule (sLe); ganglioside GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; high molecularweight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside(OAcGD2); Folate receptor alpha; Folate receptor beta; tumor endothelialmarker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R);claudin 6 (CLDN6); G protein-coupled receptor class C group 5, member D(GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a;anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1(PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH);mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2);Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3(ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2(OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumorprotein (WTI); ETS translocation-variant gene 6, located on chromosome12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A(XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); CT(cancer/testis (antigen)); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53;p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin B1; Cyclin DI; v-myc avian myelocytomatosisviral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog FamilyMember C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor(Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma AntigenRecognized By T Cells-1 or 3 (SART1, SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint-1, -2, -3 or -4 (SSX1, SSX2, SSX3, SSX4);CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1(LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300molecule-like family member f (CD300LF); C-type lectin domain family 12member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyteantigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); mousedouble minute 2 homolog (MDM2); livin; alphafetoprotein (AFP);transmembrane activator and CAML Interactor (TACI); B-cell activatingfactor receptor (BAFF-R); V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog (KRAS); immunoglobulin lambda-like polypeptide 1 (IGLL1); 707-AP(707 alanine proline); ART-4 (adenocarcinoma antigen recognized by T4cells); BAGE (B antigen; b-catenin/m, b-catenin/mutated); CAMEL(CTL-recognized antigen on melanoma); CAPi (carcinoembryonic antigenpeptide 1); CASP-8 (caspase-8); CDC27m (cell-division cycle 27 mutated);CDK4/m (cycline-dependent kinase 4 mutated); Cyp-B (cyclophilin B); DAM(differentiation antigen melanoma); EGP-2 (epithelial glycoprotein 2);EGP-40 (epithelial glycoprotein 40); Erbb2, 3, 4 (erythroblasticleukemia viral oncogene homolog-2, -3, 4); FBP (folate binding protein);, fAchR (Fetal acetylcholine receptor); G250 (glycoprotein 250); GAGE (Gantigen); GnT-V (N-acetylglucosaminyltransferase V); HAGE (helicoseantigen); ULA-A (human leukocyte antigen-A); HST2 (human signet ringtumor 2); KIAA0205; KDR (kinase insert domain receptor); LDLR/FUT (lowdensity lipid receptor/GDP L-fucose; b-D-galactosidase 2-a-Lfucosyltransferase); L1CAM (L1 cell adhesion molecule); MC1R(melanocortin 1 receptor); Myosin/m (myosin mutated); MUM-1, -2, -3(melanoma ubiquitous mutated 1, 2, 3); NA88-A (NA cDNA clone of patientM88); KG2D (Natural killer group 2, member D) ligands; oncofetal antigen(h5T4); p190 minor bcr-abl (protein of 190KD bcr-abl); Pml/RARa(promyelocytic leukaemia/retinoic acid receptor a); PRAME(preferentially expressed antigen of melanoma); SAGE (sarcoma antigen);TEL/AML1 (translocation Ets-family leukemia/acute myeloid leukemia 1);TPI/m (triosephosphate isomerase mutated); CD70; and any combinationthereof.
 39. (canceled)
 40. The T cell according to claim 29, whereinthe T cell is further modified to comprise decreased expression oractivity of, or modified to comprise an agent capable of decreasingexpression or activity of a gene or polypeptide selected from the groupconsisting of TOB1, RGS1, TARP, NKG7, CCL4 and any combination thereof,and/or wherein the T cell is further modified to comprise decreasedexpression or activity of the T cell receptor alpha constant chain locus(TRAC): and/or wherein the T cell is activated: and/or wherein the Tcell is modified using a CRISPR system comprising guide sequencesspecific to the target, preferably, wherein the CRISPR system comprisesCas9, Cas12, Cas13, or Cas14.
 41. (canceled)
 42. (canceled) 43.(canceled)
 44. (canceled)
 45. A population of T cells comprising T cellsaccording to claim 29, preferably, wherein the population of T cells isa pharmaceutical composition.
 46. (canceled)
 47. A method of treatingcancer in a subject in need thereof comprising administering thepharmaceutical composition according to claim 45 to the subject.
 48. Themethod of treatment according to claim 47, wherein the population ofcells are administered by infusion into the cerebral spinal fluid (CSF),pleural cavity or peritoneal cavity; or wherein the population of cellsare administered by injection into the cerebral spinal fluid (CSF)through the lateral ventricle.
 49. (canceled)
 50. The method oftreatment according to 47, wherein the population of cells areadministered in a combination treatment regimen comprising checkpointblockade therapy, preferably, wherein the checkpoint blockade therapycomprises anti-PD-1, anti-CTLA4, anti-PDL1, anti-TIM-3 and/or anti-LAG3.51. (canceled)
 52. The method according to 47, wherein the cancerexpresses CLEC2D; and/or wherein tumor infiltrating lymphocytes (TILs)in the cancer express KLRB1; and/or wherein the cancer is glioblastomamultiforme (GBM).
 53. (canceled)
 54. (canceled)
 55. The method of claim1, wherein administration of the one or more agents is based on firstdetermining if a sample obtained from the disease compartment of thesubject is characterized by increased expression of KLRB1, CD161, and/orCLEC2D as compared to a control.